Phosphinoamidite carboxylates and analogs thereof in the synthesis of oligonucleotides having reduced internucleotide charge

ABSTRACT

Nucleoside phosphinoamidite carboxylates and analogs are provided that have the structure of formula (III)  
                 
 
wherein A is hydrogen, hydroxyl, lower alkoxy, lower alkoxy-substituted lower alkoxy, halogen, SH, NH 2 , azide or DL wherein D is O, S or NH and L is a heteroatom-protecting group, unsubstituted hydrocarbyl, substituted hydrocarbyl, heteroatom-containing hydrocarbyl, or substituted heteroatom-containing hydrocarbyl; B is a nucleobase; and one of R 11  and R 12  is a blocking group and the other is (IV) or (VI)  
                 
 
in which W, X, Y, Z, R 1  and n are as defined herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional application of U.S. Ser. No. 10/721,301, filed Nov.24, 2003, which is a divisional application of U.S. Ser. No. 09/691,824,filed Oct. 17, 2000, now U.S. Pat. No. 6,693,187. The disclosures of theaforementioned applications are incorporated by reference.

TECHNICAL FIELD

This invention relates generally to the fields of nucleic acid chemistryand oligonucleotide synthesis, and more particularly relates to novelphosphinoamidite carboxylates and analogs thereof in the synthesis ofoligonucleotides having reduced internucleotide charge and enhancednuclease resistance, i.e., phosphinocarboxylate oligonucleotides,phosphonocarboxylate oligonucleotides, and analogs thereof.

BACKGROUND

The derivatives of phosphoric acid have been shown to have a wide rangeof biological utility (Emsley and Hall (1976) in The Chemistry ofPhosphorus: Chapter 12 Biophosphorus Chemistry pp. 471-510 Harper andRow: London, England). In turn, molecules that mimic phosphoric acid andits derivatives have been shown to work as biological effectors and areoften used as diagnostic and therapeutic agents (Uhlmann and Peyman(1990) Chem. Rev. 90: 544). Examples of these derivatives arephosphonocarboxylates (Becker et al. (1977) Antimicrob. AgentsChemother. 11: 919), phosphorothioates (Eckstein (1989) Trends Biochem.Sci. 14: 97), phosphorodithioates (Nielsen et al. (1988) TetrahedronLett. 29: 2911), methylphosphonates (Miller and Ts'o (1988) Annu. Rep.Med. Chem. 23: 295), and phosphoramidates (Iyer et al. (1996)Tetrahedron Lett. 37: 1543).

Phosphonocarboxylate mimics of phosphoric acid, specificallyphosphonoformic acid and phosphonoacetic acid, have been shown to beespecially useful as biological effectors and have been used astherapeutic agents (Shipkowitz et al. (1973) Appl. Microbiol. 26: 264;Helgestrand et al. (1978) Science 201: 819). The syntheses ofphosphonoformic acid (Nylen (1924) Chem Berichte. 57: 1023) andphosphonoacetic acid (Basinger et al. (1959) J. Org. Chem. 24: 434) haverelied upon the introduction of the carboxylate group onto thephosphorus moiety through an oxidative transformation such as aMichaelis-Arbuzov reaction (Arbuzov and Dunin (1914) J. Chem. Soc. 653;Arbuzov (1964) Pure Appl. Chem. 9: 307). The resultingphosphonocarboxylic acid products are in the oxidation state P(V). Oncethe phosphorus atom is in this pentacoordinate oxidation state theproducts are typically very stable. However, these stable products aredifficult and sometimes impossible to utilize in performing highyielding chemical transformations, chemical couplings, or chemicalderivatizations. As a result of the low chemical reactivity of thesepentacoordinate phosphorus molecules, many biologically importantmolecules that exist as phosphoric acid derivatives have not beenmimicked with phosphonocarboxylic acid derivatives (Hildebrand (1983),in The Role of Phosphonates in Living Systems: Chapters 5 & 6, pp.97-169, CRC Press Inc: Boca Raton, USA).

Two clear examples of biologically important molecules that existnaturally as phosphoric acid derivatives and have not been mimicked asphosphonocarboxylic acid derivatives are the polynucleotides DNA andRNA. Polynucleotides modified at the phosphodiester internucleotidelinkage are of significant interest to the emerging fields of antisensetherapeutics, nucleic acid diagnostics, and genomics.Phosphorus-containing chemical compounds and compositions that have beensuccessfully utilized to enable the synthesis of polynucleotides havebeen frequently reviewed in the scientific literature (Verma et al.(1998) Annu. Rev. Biochem. 67:99; Sekine et. al. (1998) Nucleosides andNucleotides 17:2033; Iyer et al (1999) Curr. Opin. Mol. Ther. 1:344).The successful chemical synthesis of polynucleotides or modifiedpolynucleotides is a task especially dependent upon the ability to findand employ phosphorus-containing compounds that enable high yieldchemical couplings and chemical transformations (Caruthers (1985)Science 230:281; Caruthers et al., U.S. Pat. No. 4,415,732, issued Nov.15, 1983). To enable the chemical synthesis of polynucleotides ormodified polynucleotides, the phosphorus compounds used must be able toperform high yield coupling reactions that are general to the fournucleobases and specific for the desired polynucleotide products. Highyield coupling efficiencies for the formation of internucleotide bondsare necessary in order to enable the synthesis of biologically relevantlengths of polynucleotides (Koster et al., U.S. Pat. No. 4,725,677issued Feb. 16, 1988), wherein a “biologically relevant length” is alength that allows the polynucleotide to stably and specifically bind toother polynucleotides by hybridization through base-pairinginteractions. Stable binding of polynucleotides to other polynucleotidesvia hybridization is also affected by temperature, salt concentration,nucleotide sequence, and other factors, as has been extensivelydiscussed in the literature; see, e.g., Sanger (1984) in Principles ofNucleic Acid Structure: Chapter 6, pp. 116-158 (Springer-Verlag: NewYork, USA).

The need for high yield coupling reactions in synthesizingpolynucleotides of a biologically relevant length is due to themathematical relationship between the final yield of the desiredpolynucleotide product and the efficiency for each individual couplingreaction giving rise to a new internucleotide bond. The final yield ofthe desired polynucleotide product is a multiplication product of allindividual coupling and deprotection steps required in achieving thatproduct. As a result, the yield of the final polynucleotide productdecreases exponentially with a linear decrease in the couplingefficiency. That is, the effect of the coupling efficiency on theoverall yield of product can be described by the equation Y=X^(N), whereY is the fractional overall yield, X is the fractional couplingefficiency, and N is the number of couplings. For the synthesis of atypical polynucleotide 20 nucleotides in length with 19 internucleotidelinkages, 19 coupling reactions are involved and the overall yield isgiven by Y=X¹⁹. The table below illustrates the relationship between thecoupling efficiency (X) and overall yield of polynucleotide product (Y).X 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 0.95 0.99 Y 1⁻¹⁹ 5⁻¹⁴1⁻¹⁰ 3⁻³ 2⁻⁶ 6⁻⁵ 1⁻³ 1⁻² 0.14 0.38 0.82

As clearly illustrated by this example, and as well known by thoseskilled in the art, the synthesis of a 20-mer polynucleotide is notpossible until the coupling efficiency achieved during synthesisapproaches 90% or greater. Only at these coupling efficiencies canfull-length polynucleotides be reproducibly isolated and purified fromthe reaction mixtures. As a further example for illustration, at an 80%coupling efficiency, the theoretical maximum amount of full-lengthproduct (Y), after 19 couplings, is 1.4%. However, the overall yieldshown in the table above is a simplification that considers only theeffect of the efficiency for the formation of the internucleotide bond.The actual overall yield of a polynucleotide product is additionallyadversely affected by any inefficiency in deprotection reactions usedduring synthesis, post-synthesis, or from side-reactions leading toundesired products. The ability to isolate a full-length polynucleotideproduct, 20 nucleotides in length, from a polynucleotide synthesis thatachieves an 80% per cycle coupling efficiency is precarious and rarelyreproducible, and directly linked to the yield of the individualdeprotection reactions following each coupling step. As a direct resultof these requirements for high yield reactions, the chemical synthesisof polynucleotides has been accomplished by only very few methods; seeBrown (1983) in Protocols for Oligonucleotides and Analogs: Chapters 1,pp. 1-17 (Humana Press: Totowa, N.J., USA, Ed. S. Agrawal). Each of themethods that has enabled the chemical synthesis of polynucleotides hasin turn been enabled by the development of high yielding couplingreactions at the phosphorus moiety, in concert with the development ofhigh yielding deprotection reactions. The fact that so few methods haveenabled polynucleotide synthesis is a direct result of the difficulty ofperforming a long series of sequential chemical reactions inquantitative or near-quantitative yields.

The chemical synthesis of polynucleotides containing the naturallyoccurring phosphodiester linkage was originally accomplished usingnucleotide building blocks known as “diester intermediates.” These“diester” building blocks were nucleotide monomers on which theheterocyclic bases and exposed hydroxyl functionality were chemicallyprotected by blocking groups, and the pendant phosphate group activatedfor transesterification reactions by the formation of a “phosphodiester”(Gilham & Khorana (1958) J. Amer. Chem Soc. 80: 6212). The lowreactivity of these activated P(V) monomers resulted in low syntheticyields for internucleotide bond formation. The typical yield for thecoupling of nucleoside phosphodiester monomers to nucleoside ornucleotide hydroxyl groups is in the range of 20-50%. The low syntheticyields achieved by these chemical coupling reactions limited this methodto the synthesis of monomer, dimer, and trimer products. The synthesisof biologically relevant lengths of polynucleotides from these monomer,dimer, and trimer blocks was accomplished by the use of an enzymaticligation reaction (Khorana (1966) The Harvey Lectures, ser.62, pp.79-106; Khorana, H. G. (1979) Science 203: 614). This enzymatic processwas dependent upon these monomer, dimer, and trimer blocks acting ashigh yielding substrates for enzymatic ligation reactions, and on theenzymatic reactions producing the desired naturally occurringphosphodiester internucleotide bond. The complete chemical synthesis ofthese polynucleotides could not be demonstrated as a result of the lowcoupling efficiencies of phosphodiester intermediates for the formationof internucleotide bonds.

The complete chemical synthesis of polynucleotides containing naturallyoccurring phosphodiester internucleotide linkages was first madepossible by the development of phosphotriester reactive intermediates(Narang et. al. (1980) Methods in Enzymology 65:610) and phosphitereactive intermediates (Letsinger & Lunsford (1976) J. Am. Chem. Soc.98:3655; Beaucage & Caruthers (1981) Tetrahedron Lett. 22:1859).Phosphotriester reactive intermediates produced coupling efficienciesfor the formation of internucleotide bonds in the range of 65-87%depending upon the sequence and condensing agent utilized (Efimov et.al. (1982) Nucleic Acids Research 10:6675). In the lower end of thisrange of coupling efficiencies, the enzymatic ligation of block-coupledproducts was required to enable the synthesis and isolation ofpolynucleotides. In the upper end of the range of coupling efficiencies,it was difficult but possible to isolate full-length polynucleotidesfrom the reaction products of a complete chemical synthesis. Thesubsequent development of phosphite reactive intermediates producedcoupling efficiencies for the formation of internucleotide bonds in therange of 90-99%. It was the invention of these phosphite chemicalcompositions of protected nucleosides, in concert with effectiveprotecting group chemistry, that enabled the routine chemical synthesisof native polynucleotides (Matteucci & Caruthers (1981) J. Am. Chem.Soc. 103: 3185).

The chemical synthesis of phosphorus-backbone modified polynucleotidesis more difficult than the chemical synthesis of naturally occurringphosphodiester-linked polynucleotides. Modification of the phosphorusbackbone, in most cases, precludes the use of high yielding enzymaticligation methods. The enzymes that are typically used for the formationof internucleotide bonds have substrate fidelity requirements that makeit difficult if not impossible to use them to form backbone-modifiedinternucleotide bonds. A few phosphorus backbone modifications, such asphosphorothioates, have been formed using enzymatic incorporation ofmodified nucleotides or enzymatic ligation reactions, but thesemodifications tend to be rare and the reactions much less efficient thanthe formation of native internucleotide bonds (Eckstein (1985) Annu.Rev. Biochem. 54:367). Without the ability to overcome low couplingefficiencies using enzymatic ligation of dimer and trimer blocks, theenablement of backbone-modified polynucleotides is even more rigorouslytied to the need for high yielding coupling reactions and high yieldingdeprotection reactions than is the synthesis of phosphodiester-linkedpolynucleotides.

Nucleotide monomers of phosphonocarboxylates, phosphonoformic acid andphosphonoacetic acid (Heimer et. al., U.S. Pat. No. 4,056,673 issuedNov. 1, 1977; Sekine et. al. (1982) Bull. Chem. Soc. Jpn. 55: 239;Griengl et. al. (1988) J. Med. Chem. 31: 1831; Lambert et. al. (1989) J.Med Chem. 32: 367) have been prepared using protected nucleosides andthe activated transesterification techniques developed forphosphodiester and phosphotriester coupling of internucleotide bonds(Shaller et. al. (1963) J. Am. Chem. Soc. 85: 3821; Amarnath et al.(1977) Chem. Rev. 77: 183). These modified nucleotide monomers have beenshown to be inhibitory to DNA and RNA polymerases and have not proved tobe efficient substrates for enzymatic incorporation or ligation. As aresult, the enablement of phosphonocarboxylate derivatives ofpolynucleotides requires the ability to perform complete chemicalsynthesis, in turn requiring high yielding coupling reactions and highyielding deprotection reactions.

Prior to the current invention, attempts by the present inventor tochemically synthesize phosphonocarboxylate modified polynucleotides wereperformed using the chemical reactions previously reported for couplingalkyl esters of phosphonoformic acid and phosphonoacetic acid toprotected nucleosides. 3′-Phosphonocarboxylate protected nucleotidemonomers were prepared and isolated by literature protocols. A series ofattempts were then made to produce polynucleotides withphosphonocarboxylate-modified internucleotide linkages by applying oneof the many standard phosphotriester condensing agents on solid-phase(Stawinski et. al. (1977) Nucleic Acids Res. 4: 353; Reese et. al.(1978) Tetrahedron Lett. 19: 2727). All of these attempts to forminternucleotide bonds using activated transesterification methods on theP(V) phosphonocarboxylates of protected nucleosides, gave couplingefficiencies too poor to enable the synthesis of polynucleotides.Although the coupling efficiencies were extremely low (<10%),solution-phase coupling of these modified nucleotides to 3′-protectednucleosides allowed for isolation of a small amount ofthymidine-thymidylate protected dimers with phosphonoformate andphosphonoacetate internucleotide bonds. Attempts to deprotect the ethylor methyl esters of the carboxylic acid phosphonate dimers, by thehydrolysis methods previously published for nucleotidemonophosphonocarboxylates, led to cleavage of the internucleotide bond.Using base-catalyzed, nucleophilic hydrolysis conditions to deprotectthe carboxylic acid methyl or ethyl esters of phosphonoformate andphosphonoacetate thymidine-thymidylate dimers, resulted in significantcleavage of the phosphorus-carbon bond (40%-100%). Analysis of theproducts from these nucleophilic hydrolysis reactions demonstratedcleavage of the phosphorus-carbon bond that in turn resulted in bothcleavage of the internucleotide bond, and conversion of the modifiedinternucleotide bond to a phosphodiester bond. A subsequent literaturestudy confirmed these observations and revealed that thephosphorus-carbon bond of acylphosphonates was susceptible to cleavageunder the conditions of carboxylate ester hydrolysis. The facile loss ofsimilar phosphorus-carbon bonds had been previously observed under theconditions of nucleophilic hydrolysis and a mechanism proposed (Sekineet al. (1980) J. Org. Chem. 45: 4162; Narayanan et. al (1979) J. Am.Chem. Soc. 101: 109; Kluger et. al. (1978) J. Am. Chem. Soc. 100: 7382).The nucleophilic attack of hydroxide or other nucleophile on thecarbonyl of phosphonocarboxylates result in the formation of atetrahedral intermediate containing a localized negative charge on theoxygen of the carbonyl. Further rearrangement of this intermediateresults in reaction products that favor cleavage of thephosphorus-carbon bond rather than the desired substitution byhydroxide. Once again, there is a high yield requirement for thesedeprotection reactions in order to enable the synthesis and isolation ofphosphonocarboxylic acid modified polynucleotides. Non-quantitativedeprotection reactions at the internucleotide linkage or rearrangementproducts that lead to undesired side-products has an exponentiallynegative effect on the yield of full-length polynucleotide product.Moderate to low yields for the removal of protecting groups, or cleavageof the phosphorus-carbon bond during deprotection of the carboxylic acidthus directly prevents the enablement of these modified polynucleotides.Although one reference describes synthesis of phosphonocarboxylic acidmodified polynucleotides as feasible (Cook et al., International PatentPublication No. WO 93/10140), in fact the methods described, or similaractivated transesterification methods, do not enable the synthesis ofphosphonocarboxylic acid modified polynucleotides.

Rudolph et. al (1996) Nucleosides and Nucleotides 15:1725 describe thesolution-phase synthesis of thymidine-thymidylate dimers containingmethyl phosphonoacetate internucleotide bonds using an activatedtransesterification method with1-(2-mesitylene-sulfonyl)-3-nitro-1,2,4-triazole (MSNT) and apentavalent phosphonoacetate derivative. In order to incorporate thisdimer modification into longer polymers the authors derivatized thedimer with a standard phosphoramidite reagent (Atkinson et al. (1984)Oligonucleotide Synthesis: A Practical Approach, Gait, Ed., IRL Press,Oxford, pp 41-45) and used the high yielding formation of nativeinternucleotide phosphodiester bonds to assemble longer polymers,containing only the nucleobase thymidine, with the assumption that everyother linkage in the resulting reaction mixture would be aphosphonoacetate. The publication additionally reported that thephosphonoacetate group is labile to ammonium hydroxide, hydrazine,ethylenediamine, triethylamine/water, and piperidine/water, resulting inthe cleavage of the internucleotide bond. The conditions reported togive complete cleavage of the internucleotide bond are the sameconditions that are reported in WO 93/10140. As in the aforementionedPCT publication, Rudolph et al. clearly demonstrated that the activatedtransesterification coupling of protected nucleoside, P(V) alkylphosphonocarboxylates and subsequent hydrolytic cleavage of alkyl esterprotected carboxylic acid groups does not enable the synthesis ofphosphonocarboxylic acid modified polynucleotides.

There are few reported phosphorus chemical compositions in the oxidationstate P(III) having protected carboxylic acid functional groups. Reportsof phosphinylacetic acid derivatives are uncommon, and have beenexclusively studied for their unique physical (Matrosov et. al. (1972)Zh. Obshch. Khim. 42: 1695) and chemical (Podlahova, J. (1978)Collection Czechoslov. Chem. Commun. 43: 57) properties rather thantheir use as chemical synthons. Also see Novikova et. al. (1976) Zh.Obshch. Khim. 46: 575; Novikova et. al. (1976) Zh. Obshch. Khim. 46:2213; and Stepanov et. al. (1979) Zh. Obshch. Khim. 49: 2389)

The low chemical reactivity of pentacoordinate phosphonocarboxylatemolecules has prevented the incorporation of internucleotidephosphonocarboxylate moieties into many biologically importantmolecules. Preparation of oligonucleotides containing internucleotidephosphonocarboxylate moieties would require chemical compositions ofphosphorus that perform high yielding chemical couplings and chemicaltransformations. More particularly, chemical synthesis ofphosphonocarboxylate oligonucleotides and polynucleotides (DNA and RNA)would require both high yielding coupling reactions at the phosphorusmoiety and high yielding deprotection reactions at the carboxylatemoiety, with the phosphorus-carboxylate moiety left intact.

SUMMARY OF THE INVENTION

Accordingly, it is a primary object of the invention to providecompounds and methods for synthesizing oligonucleotides containinginternucleotide phosphinocarboxylate and phosphonocarboxylate linkagesand analogs thereof.

It is another object of the invention to provide such compounds andmethods wherein chemical synthesis of the oligonucleotides proceeds viahigh yielding coupling reactions at the phosphorus moiety as well ashigh yielding reactions at the carboxylate moiety, with thephosphorus-carboxylate moiety left intact.

It is another object of the invention to provide phosphinocarboxylateoligonucleotides, phosphonocarboxylate oligonucleotides, and analogsthereof synthesized using the aforementioned compounds and methods.

It is an additional object of the invention to provide methods for usingthe novel phosphinocarboxylate and phosphonocarboxylateoligonucleotides.

Additional objects, advantages and novel features of the invention willbe set forth in part in the description which follows, and in part willbecome apparent to those skilled in the art upon examination of thefollowing, or may be learned by practice of the invention.

In one embodiment of the invention, then, phosphinoamidite carboxylatesand analogs thereof are provided having the structure of formula (I)

wherein: R¹ is hydrogen, a protecting group removable by an eliminationreaction, hydrocarbyl, substituted hydrocarbyl, heteroatom-containinghydrocarbyl, or substituted heteroatom-containing hydrocarbyl; R² and R³are independently selected from the group consisting of hydrocarbyl,substituted hydrocarbyl, heteroatom-containing hydrocarbyl andsubstituted heteroatom-containing hydrocarbyl, or R² and R³ are linkedto form a substituted or unsubstituted, five- or six-memberednitrogen-containing heterocycle; R⁴ is NR⁵R⁶, halogen or DL, wherein R⁵and R⁶ are independently selected from the group consisting ofhydrocarbyl, substituted hydrocarbyl, heteroatom-containing hydrocarbyland substituted heteroatom-containing hydrocarbyl, or R⁵ and R⁶ arelinked to form a substituted or unsubstituted, five- or six-memberednitrogen-containing heterocycle, D is O, S or NH, and L is aheteroatom-protecting group, unsubstituted hydrocarbyl, substitutedhydrocarbyl, heteroatom-containing hydrocarbyl, or substitutedheteroatom-containing hydrocarbyl; X is O, S NH or NR⁷ wherein R⁷ ishydrocarbyl, substituted hydrocarbyl, heteroatom-containing hydrocarbylor substituted heteroatom-containing hydrocarbyl; n is zero or 1; Y is—(Y′)_(m)—(CR⁸R⁹)— wherein m is zero or 1, Y′ is hydrocarbylene,substituted hydrocarbylene, heteroatom-containing hydrocarbylene, orsubstituted heteroatom-containing hydrocarbylene, wherein R⁸ and R⁹ areas defined for R¹, with the proviso that when n is 1, m is zero and R⁸and R⁹ are both hydrogen, then R¹ is hydrogen or a protecting groupremovable by an elimination reaction; and Z is O, S, NH or NR¹⁰ whereinR¹⁰ is as defined for R⁷.

Compounds of formula (I) are useful as phosphitylating agents, in thatthe NR²R³ and/or the R⁴ moieties are leaving groups susceptible todisplacement by nucleophilic attack, e.g., by a hydroxyl or other group.For example, the compounds can be used to phosphitylate a nucleoside byreaction of a 3′-hydroxyl group or a 5′-hydroxyl group. The compoundsare also useful for phosphitylating peptides and proteins, insofar asthe compounds can phosphitylate an amino acid at any nucleophilic site,e.g., at the hydroxyl groups of serine, threonine and tyrosine, or atthe sulfhydryl group of cysteine. Surprisingly, these compounds enablephosphitylation in very high yield. In one important application, thecompounds provide the capability of sequentially synthesizing DNAoligonucleotides with very high coupling yields for each individualcoupling reaction. The compounds of formula (I) may also be useful astherapeutic agents, and may be screened for activity using conventionaltechniques.

In another embodiment, modified nucleosides are provided having aphosphinoamidite carboxylate substituent, an H-phosphonite carboxylatesubstituent, or an analog thereof at either the 3′ or the 5′ position.The modified nucleosides have the structural formula (III)

wherein: A is hydrogen, hydroxyl, lower alkoxy, lower alkoxy-substitutedlower alkoxy, halogen, SH, NH₂, azide or DL wherein D is O, S or NH andL is a heteroatom-protecting group, unsubstituted hydrocarbyl,substituted hydrocarbyl, heteroatom-containing hydrocarbyl, orsubstituted heteroatom-containing hydrocarbyl; B is a nucleobase; andone of R¹¹ and R¹² is a blocking group and the other is (IV) or (VI)

in which W is NR²R³, NR⁵R⁶ or DL, and R¹, X, Y, Z and n are as definedas follows: R¹ is hydrogen, a protecting group removable by anelimination reaction, hydrocarbyl, substituted hydrocarbyl,heteroatom-containing hydrocarbyl, or substituted heteroatom-containinghydrocarbyl; R² and R³ are independently selected from the groupconsisting of hydrocarbyl, substituted hydrocarbyl,heteroatom-containing hydrocarbyl and substituted heteroatom-containinghydrocarbyl, or R² and R³ are linked to form a substituted orunsubstituted, five- or six-membered nitrogen-containing heterocycle; R⁴is NR⁵R⁶, halogen or DL, wherein R⁵ and R⁶ are independently selectedfrom the group consisting of hydrocarbyl, substituted hydrocarbyl,heteroatom-containing hydrocarbyl and substituted heteroatom-containinghydrocarbyl, or R⁵ and R⁶ are linked to form a substituted orunsubstituted, five- or six-membered nitrogen-containing heterocycle, Dis O, S or NH, and L is a heteroatom-protecting group, unsubstitutedhydrocarbyl, substituted hydrocarbyl, heteroatom-containing hydrocarbyl,or substituted heteroatom-containing hydrocarbyl; X is O, S NH or NR⁷wherein R⁷ is hydrocarbyl, substituted hydrocarbyl,heteroatom-containing hydrocarbyl or substituted heteroatom-containinghydrocarbyl; n is zero or 1; Y is —(Y′)_(m)—(CR⁸R⁹)— wherein m is zeroor 1, Y′ is hydrocarbylene, substituted hydrocarbylene,heteroatom-containing hydrocarbylene, or substitutedheteroatom-containing hydrocarbylene, wherein R⁸ and R⁹ are as definedfor R¹; and Z is O, S, NH or NR¹⁰ wherein R¹⁰ is as defined for R⁷.

When the nucleoside contains moiety (IV), it is a nucleosidephosphinoamidite carboxylate or analog thereof, while when thenucleoside contains moiety (VI), it is a nucleoside H-phosphonitecarboxylate or analog thereof.

In still another embodiment, modified oligonucleotides are provided thathave reduced internucleotide charge at physiological pH and enhancedstability to degradation by nucleases. The modified oligonucleotidescontain at least one internucleotide linkage having the structure offormula (VIII) or (X)

in which R¹, X, Y, Z and n are as defined above with respect to themodified nucleosides of formula (III), wherein when the internucleotidelinkage is (VIII), the oligonucleotide is a “phosphinocarboxylate”oligonucleotide or analog thereof, while when the internucleotidelinkage is (X), the oligonucleotide is a “phosphonocarboxylate”oligonucleotide or analog thereof. These phosphinocarboxylate andphosphonocarboxylate oligonucleotides find applications in a variety ofareas, for example: as therapeutic agents, particularly as therapeuticagents in antisense-, ribozyme- and aptamer-based strategies; asdiagnostic agents in target validation, to test selected proteins forsuitability as a therapeutic target; in investigating the mechanism andstereochemistry of biochemical reactions; and in the mapping of nucleicacid protein interactions. Furthermore, since the novel oligonucleotideshave unexpectedly been found to direct the hydrolysis of complementaryRNA in the presence of RNaseH, they are also useful as agents foreliciting RNaseH activity. Finally, because the oligonucleotides havereduced charge relative to relative to oligonucleotides with standardphosphonate linkages, they readily pass through body membranes and inaddition have enhanced utility in the area of mass spectrometry.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically illustrates synthesis of phosphonoacetatepolynucleotides on a solid support, as described in detail in Example33.

FIG. 2 schematically illustrates synthesis of phosphonothioacetatepolynucleotides on a solid support, as described in detail in Example34.

FIGS. 3 and 4 illustrate the results of ion exchange HPLC of 18-mermixed sequence phosphonoacetate and phosphonothioacetateoligonucleotides, respectively, as described in Example 36.

FIGS. 5 and 6 provide the results of matrix-assisted laser desorptionionization time of flight (MALDI-TOF) spectroscopic analysis on mixedsequence 18-mer phosphonoacetate and phosphonothioacetateoligonucleotides, respectively, as described in detail in Example 37.

FIG. 7 is an autoradiogram of ³²P-5′-end labeled oligodeoxynucleotide18-mers in the presence of snake venom phosphodiesterase, as describedin Example 38.

FIG. 8 is an autoradiogram of duplexes of ³²P-5′-end labeledoligodeoxynucleotide 18-mers in the presence of DNaseI, as described inExample 38.

FIG. 9 is an autoradiogram of ³²P-5′-end labeled RNA in the presence ofcomplementary oligonucleotides and E. coli RNaseH1, as described inExample 39.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions and Overview

Before describing the present invention in detail, it is to beunderstood that unless otherwise indicated this invention is not limitedto specific compounds, reagents, reaction conditions, synthetic steps,or the like, as such may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a protecting group” includes combinations of protectinggroups, reference to “a nucleoside” includes combinations ofnucleosides, and the like. Similarly, reference to “a substituent” as ina compound substituted with “a substituent” includes the possibility ofsubstitution with more than one substituent, wherein the substituentsmay be the same or different.

As used herein the symbols for nucleosides, nucleotides andpolynucleotides are according to the IUPAC-IUB Commission of BiochemicalNomenclature recommendations (Biochemistry 9:4022 (1970)).

In this specification and in the claims which follow, reference will bemade to a number of terms which shall be defined to have the followingmeanings:

As used herein, the phrase “having the structure” is not intended to belimiting and is used in the same way that the term “comprising” iscommonly used. The term “independently selected from the groupconsisting of” is used herein to indicate that the recited elements,e.g., R groups or the like, can be identical or different.

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances where itdoes not. For example, the phrase “optionally substituted hydrocarbyl”means that a hydrocarbyl moiety may or may not be substituted and thatthe description includes both unsubstituted hydrocarbyl and hydrocarbylwhere there is substitution.

The term “alkyl” as used herein refers to a branched or unbranchedsaturated hydrocarbon group typically although not necessarilycontaining 1 to about 20 carbon atoms, such as methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl, and the like, aswell as cycloalkyl groups such as cyclopentyl, cyclohexyl and the like.Generally, although again not necessarily, alkyl groups herein contain 1to about 12 carbon atoms. The term “lower alkyl” intends an alkyl groupof 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms. “Substitutedalkyl” refers to alkyl substituted with one or more substituent groups,and the terms “heteroatom-containing alkyl” and “heteroalkyl” refer toalkyl in which at least one carbon atom is replaced with a heteroatom.The term “cycloalkylalkyl” refers to an alkyl group substituted with acycloalkyl substituent.

The term “alkylene” as used herein refers to difunctional, branched orunbranched saturated hydrocarbon group typically although notnecessarily containing 1 to about 20 carbon atoms, such as methylene,ethylene, n-propylene, n-hexylene, methylethylene, and the like.Generally, although again not necessarily, alkylene groups hereincontain 1 to about 12 carbon atoms. The term “lower alkylene” intends analkylene group of 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms.“Substituted alkylene” refers to alkylene substituted with one or moresubstituent groups, and the terms “heteroatom-containing alkylene” and“heteroalkylene” refer to alkylene in which at least one carbon atom isreplaced with a heteroatom.

The term “alkenyl” as used herein refers to a branched or unbranchedhydrocarbon group typically although not necessarily containing 2 toabout 20 carbon atoms and at least one double bond, such as ethenyl,n-propenyl, isopropenyl, s-propenyl, 2-propenyl, n-butenyl, isobutenyl,octenyl, decenyl, and the like. Generally, although again notnecessarily, alkenyl groups herein contain 3 to about 10 carbon atoms.The term “lower alkenyl” intends an alkenyl group of 2 to 6 carbonatoms, preferably 2 to 4 carbon atoms. “Substituted alkenyl” refers toalkenyl substituted with one or more substituent groups, and the terms“heteroatom-containing alkenyl” and “heteroalkenyl” refer to alkenyl inwhich at least one carbon atom is replaced with a heteroatom.

The term “alkynyl” as used herein refers to a branched or unbranchedhydrocarbon group typically although not necessarily containing 2 toabout 20 carbon atoms and at least one triple bond, such as ethynyl,n-propynyl, n-butynyl, octynyl, decynyl, and the like. Generally,although again not necessarily, alkynyl groups herein contain 3 to about10 carbon atoms. The term “lower alkynyl” intends an alkynyl group of 2to 6 carbon atoms, preferably 2, 3 or 4 carbon atoms. “Substitutedalkynyl” refers to alkynyl substituted with one or more substituentgroups, and the terms “heteroatom-containing alkynyl” and“heteroalkynyl” refer to alkynyl in which at least one carbon atom isreplaced with a heteroatom.

The term “alkoxy” as used herein intends an alkyl group bound through asingle, terminal ether linkage; that is, an “alkoxy” group may berepresented as —O-alkyl where alkyl is as defined above. A “loweralkoxy” group intends an alkoxy group containing 1 to 6, more preferably1 to 4, carbon atoms.

The term “amino” is used herein to refer to the —NH₂ group, while“substituted amino” refers to —NHZ¹ and —NZ¹ Z² groups, where each of Z¹and Z² is independently selected from the group consisting of optionallysubstituted hydrocarbyl and heteroatom-containing hydrocarbyl, orwherein, in disubstituted amino groups, Z¹ and Z² may be linked to forman optionally substituted hydrocarbylene or heteroatom-containinghydrocarbylene bridge.

The term “aryl” as used herein, and unless otherwise specified, refersto an aromatic substituent containing a single aromatic ring or multiplearomatic rings that are fused together, linked covalently, or linked toa common group such as a methylene or ethylene moiety. The commonlinking group may also be a carbonyl as in benzophenone, an oxygen atomas in diphenylether, or a nitrogen atom as in diphenylamine. Preferredaryl groups contain one aromatic ring or two fused or linked aromaticrings, e.g., phenyl, naphthyl, biphenyl, diphenylether, diphenylamine,benzophenone, and the like. “Substituted aryl” refers to an aryl moietysubstituted with one or more substituent groups, and the terms“heteroatom-containing aryl” and “heteroaryl” refer to aryl in which atleast one carbon atom is replaced with a heteroatom.

The term “arylene” as used herein, and unless otherwise specified,refers to a divalent aromatic substituent containing a single aromaticring or multiple aromatic rings that are fused together or linkedcovalently. Preferred arylene groups contain one aromatic ring or twofused or linked aromatic rings. “Substituted arylene” refers to anarylene moiety substituted with one or more substituent groups, and theterms “heteroatom-containing arylene” and “heteroarylene” refer toarylene in which at least one carbon atom is replaced with a heteroatom.

The term “aralkyl” refers to an alkyl group with an aryl substituent,the term “aralkenyl” refers to an alkenyl group with an arylsubstituent, the term “aralkynyl” refers to an alkynyl group with anaryl substituent, and the term “aralkylene” refers to an alkylene groupwith an aryl substituent. The term “alkaryl” refers to an aryl groupthat has an alkyl substituent, the term “cycloalkylaryl” refers to anaryl group that has a cycloalkyl substituent, and the term “alkarylene”refers to an arylene group with an alkyl substituent.

The terms “halo” and “halogen” are used in the conventional sense torefer to a chloro, bromo, fluoro or iodo substituent. The terms“haloalkyl,” “haloalkenyl” or “haloalkynyl” (or “halogenated alkyl,”“halogenated alkenyl,” “halogenated aromatic” or “halogenated alkynyl”)refers to an alkyl, alkenyl, aromatic or alkynyl group, respectively, inwhich at least one of the hydrogen atoms in the group has been replacedwith a halogen atom.

The term “heteroatom-containing” as in a “heteroatom-containinghydrocarbyl group” refers to a molecule or molecular fragment in whichone or more carbon atoms is replaced with an atom other than carbon,e.g., nitrogen, oxygen, sulfur or phosphorus. Similarly, the term“heteroalkyl” refers to an alkyl substituent that isheteroatom-containing, the term “heterocyclic” refers to a cyclicsubstituent that is heteroatom-containing, the term “heteroaryl” refersto an aryl substituent that is heteroatom-containing, and the like.Heteroatoms can also replace certain carbon atoms as part of unsaturatedsystems such as wherein an oxygen atom replaces a carbon atom in analkene to generate a ketone or aldehyde, and wherein a nitrogen atomreplaces a carbon atom in an alkyne to generate a nitrile. Examples ofcommon heteroatom-substituted radicals used in nucleotide chemistry areβ-cyanoethyl, methyl-β-cyanoethyl, dimethyl-β-cyanoethyl,phenylsulfonylethyl, methyl-sulfonylethyl, p-nitrophenylsulfonylethyl,2,2,2-trichloro-1,1-dimethylethyl, 2-(4-pyridyl)ethyl,2-(2-pyridyl)ethyl, -thiobenzoylethyl, 1,1,1,3,3,3-hexafluoro-2-propyl,2,2,2-trichloroethyl, p-nitrophenylethyl, p-cyanophenylethyl, acetyl,tetrahydropyranyl, di-p-methoxytrityl, and benzoyl radicals. When theterm “heteroatom-containing” appears prior to a list of possibleheteroatom-containing groups, it is intended that the term apply toevery member of that group.

“Hydrocarbyl” refers to univalent hydrocarbyl radicals containing 1 toabout 30 carbon atoms, preferably 1 to about 20 carbon atoms, mostpreferably 1 to about 12 carbon atoms, including branched or unbranched,saturated or unsaturated species, such as alkyl groups, alkenyl groups,aryl groups, and the like. The term “lower hydrocarbyl” intends ahydrocarbyl group of 1 to 6 carbon atoms, preferably 1 to 4 carbonatoms. The term “hydrocarbylene” intends a divalent hydrocarbyl moietycontaining 1 to about 30 carbon atoms, preferably 1 to about 24 carbonatoms, most preferably 1 to about 12 carbon atoms, including branched orunbranched, saturated or unsaturated species, or the like. The term“lower hydrocarbylene” intends a hydrocarbylene group of one to sixcarbon atoms, preferably one to four carbon atoms. “Substitutedhydrocarbyl” refers to hydrocarbyl substituted with one or moresubstituent groups, and the terms “heteroatom-containing hydrocarbyl”and “heterohydrocarbyl” refer to hydrocarbyl in which at least onecarbon atom is replaced with a heteroatom. Similarly, “substitutedhydrocarbylene” refers to hydrocarbylene substituted with one or moresubstituent groups, and the terms “heteroatom-containing hydrocarbylene”and “heterohydrocarbylene” refer to hydrocarbylene in which at least onecarbon atom is replaced with a heteroatom.

The term “phosphoryl” as in a “phosphoryl group” refers to a pendentphosphorus-containing moiety, protected or unprotected, modified orunmodified.

The term “phosphinocarboxylate” refers to the group

while the term “phosphonocarboxylate” refers to the group

and the term “H-phosphonite carboxylate” refers to the group

wherein n, Y and R¹ are as defined previously. The term “analogs” ofphosphinocarboxylates, phosphonocarboxylates and H-phosphonitecarboxylates refers to moieties wherein either or both of the oxygenatoms in the carboxylate moiety are replaced with another atom, e.g., Sor N, and/or wherein the carboxylate is in the form of a carboxylic acid(R¹ is H) or a carboxylic acid salt in which a carboxylic acidanion_COO⁻ is associated with a cationic counterion. When the term“analog” is not explicitly used, it should be understood that“carboxylate” as used herein refers not only to the group

but also to the groups

wherein R¹, X and Z are as defined previously, and Q is a cationiccounterion that may be either organic or inorganic, e.g., a metal ion,an ammonium ion, or the like.

By “substituted” as in “substituted hydrocarbyl,” “substitutedhydrocarbylene,” “substituted alkyl,” and the like, as alluded to insome of the aforementioned definitions, is meant that in thehydrocarbyl, hydrocarbylene, alkyl, alkenyl or other moiety, at leastone hydrogen atom bound to a carbon atom is replaced with one or moresubstituents that are functional groups such as hydroxyl, alkoxy, thio,amino, halo, and the like. When the term “substituted” appears prior toa list of possible substituted groups, it is intended that the termapply to every member of that group.

It will be appreciated that, as used herein, the terms “nucleobase,”“nucleoside” and “nucleotide” refer to nucleobases, nucleosides andnucleotides containing not only the conventional purine and pyrimidinebases, i.e., adenine (A), thymine (T), cytosine (C), guanine (G) anduracil (U), but also protected forms thereof, e.g., wherein the base isprotected with a protecting group such as acetyl, difluoroacetyl,trifluoroacetyl, isobutyryl or benzoyl, and purine and pyrimidineanalogs. Where the term “nucleobase” is used, therefore, the termincludes unprotected and protected nucleosides and nucleotides oranalogs thereof. Suitable analogs will be known to those skilled in theart and are described in the pertinent texts and literature. Inaddition, the terms “nucleoside” and “nucleotide” include those moietiesthat contain not only conventional ribose and deoxyribose sugars, butother sugars as well. Modified nucleosides or nucleotides also includemodifications on the sugar moiety, e.g., wherein one or more of thehydroxyl groups are replaced with halogen atoms or aliphatic groups, orare functionalized as ethers, amines, or the like. Unless otherwiseindicated, the term “nucleoside” is also intended to encompassnucleoside monomers as well as nucleosides present within anoligonucleotide chain, either at a terminus thereof or within theoligonucleotide backbone.

As used herein, the term “oligonucleotide” shall be generic topolydeoxynucleotides (containing 2-deoxy-D-ribose), topolyribonucleotides (containing D-ribose), to any other type ofpolynucleotide which is an N-glycoside of a purine or pyrimidine base,and to other polymers containing nonnucleotidic backbones, providingthat the polymers contain nucleobases in a configuration which allowsfor base pairing and base stacking, such as is found in DNA and RNA. Theterm “oligonucleotide” includes double- and single-stranded DNA, as wellas double- and single-stranded RNA and DNA:RNA hybrids, and also includeknown types of modifications, for example, labels which are known in theart, methylation, “caps,” and the like. The oligonucleotides may benaturally occurring or chemically synthesized.

The term “protecting group” as used herein is meant a species thatprevents a segment of a molecule from undergoing a specific chemicalreaction, but which is removable from the molecule following completionof that reaction. This is in contrast to a “capping group” whichpermanently binds to a segment of a molecule to prevent any furtherchemical transformation of that segment.

The term “blocking group” as used herein is meant a species whichprevents a segment of a molecule from undergoing a specific chemicalreaction, but which may or may not be removable from the moleculefollowing completion of that reaction. An example, by way ofillustration and not limitation, of a blocking group that may or may notbe removable from the molecule is a methyl ester of a carboxylic acid. Amethyl ester of a carboxylic acid may be removed from the molecule toform an acidic functional group that under certain pH conditions givesthe molecule a negative charge, or may be left on the molecule toneutralize a resulting charge. It will be appreciated that, as usedherein, the term “blocking group” can also refer to any atom or moleculewhich may prevent a specific chemical reaction from occurring at thatsegment of the molecule. It should be noted that the term “blockinggroup” as used herein is intended to encompass “protecting groups” asdefined above. Greene and Wuts, Protective Groups in Organic Synthesis,2nd Edition (John Wiley, New York, 1991) provides extensive guidance onthe selection of removable blocking groups (i.e., protecting groups) foruse herein.

The term “electron withdrawing” denotes the tendency of a substituent toattract valence electrons of the molecule of which it is a part, i.e.,an electron-withdrawing substituent is electronegative.

The term “elimination reaction” as used herein is meant to describe achemical reaction by which a species is removed by “elimination” or“fragmentation.” This is in contrast to an operation by which a speciesis removed by a “substitution reaction.” An example of this distinctionis the contrast between the typical method for the removal of an ethylgroup from a protected carboxylic acid (i.e., an ethyl ester) using ahydroxide nucleophile, and the removal of a β-cyanoethyl blocking groupfrom a protected carboxylic acid (i.e., a β-cyanoethyl ester) using anon-nucleophilic base. The ethyl-protected carboxylic acid isdeprotected by substitution of the ethoxide group on the carbonyl groupof the ester with hydroxide, whereas the β-cyanoethyl-protectedcarboxylic acid is deprotected by elimination or fragmentation as theprotecting group is transformed by specific chemical reactions leavingthe carboxylate intact. With the β-cyanoethyl protecting group, thespecific chemical reaction is base-catalyzed β-elimination. There aremany other protecting groups, well known to those skilled in the art,which may be removed by elimination or fragmentation, leaving thecarboxylate moiety intact, rather than by substitution on the carbonyl.Examples of protecting groups that may be removed by eliminationreactions, by way of illustration and not limitation, are: β-cyanoethyl,methyl-β-cyanoethyl, dimethyl-β-cyanoethyl, phenylsulfonylethyl,methyl-sulfonylethyl, p-nitrophenylsulfonylethyl,2,2,2-trichloro-1,1-dimethylethyl, 2-(4-pyridyl)ethyl,2-(2-pyridyl)ethyl, allyl, 4-methylene-1-acetylphenol,-thiobenzoylethyl, 1,1,1,3,3,3-hexafluoro-2-propyl,2,2,2-trichloroethyl, p-nitrophenylethyl, p-cyanophenyl-ethyl,9-fluorenylmethyl, 1,3-dithionyl-2-methyl, 2-(trimethylsilyl)ethyl,2-methylthioethyl, 2-(diphenylphosphino)-ethyl, 1-methyl-1-phenylethyl,3-buten-1-yl, 4-(trimethylsilyl)-2-buten-1-yl, cinnamyl,-methylcinnamyl, and 8-quinolyl.

The terms “substrate,” “surface” and “solid phase” refer to anystructure that can be used to physically separate reactions or reactionproducts from reactants, starting materials, and/or by-products.Suitable substrate materials include, but are not limited to, supportsthat are typically used for solid phase chemical synthesis, e.g.,polymeric materials, silica and silica-based materials, glasses,ceramics, metals, and the like. In some embodiments, the substratesurface will be substantially flat although in other embodiments highlyporous materials or microbeads will be utilized.

“Optional” or “optionally” means that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.For example, recitation of a substituent as “optionally present”encompasses both the molecular moiety containing the substituent and themolecular moiety not containing the substituent.

In the molecular structures herein, single bonds are indicated in theconventional sense using a single line connecting two atoms, whiledouble bonds are indicated in the conventional sense using a double linebetween two adjacent atoms. However, it will be appreciated thatmolecular structures can be drawn in different ways, and that in somecases a particular bond may be drawn as either a single or double bond,with both representations being chemically accurate and indicating thesame structure.

It should also be emphasized that certain molecular entities ormolecular segments herein may contain one or more chiral centers andthus may be a racemic mixture (50-50) of isomers, a mixture of isomerswhere one isomer is present in excess, or a substantially pure isomer,“substantially pure” meaning that one isomer represents greater than90%, preferably greater than 95%, more preferably greater than 99%, of amixture of isomers. It is intended that for such chiral molecules thedisclosure herein encompasses a mixture of isomers as well as asubstantially pure isomer.

II. Phosphinoamidite Carboxylates and Analogs Thereof

This present invention relates in part to novel phosphorus-containingcompounds that are particularly useful for making phosphonocarboxylatemimics of naturally occurring, biologically active, nucleoside andoligonucleotide phosphonates. The novel compounds, phosphinoamiditecarboxylates and analogs thereof, are especially useful for thepreparation of protected nucleoside phosphinocarboxylates, which in turnenable formation of phosphinocarboxylate and phosphonocarboxylateinternucleotide bonds, and for the first time allow for the synthesis ofphosphinocarboxylate and phosphonocarboxylate oligonucleotides andpolynucleotides. That is, these highly reactive P(III) derivativesenable the synthesis of phosphinocarboxylate and phosphonocarboxylateoligonucleotides through the high yielding formation ofphosphinocarboxylate and phosphonocarboxylate internucleotide bonds.Generally, the phosphinoamidite carboxylates will have the carboxylatefunctional group protected with a blocking group that is removable usingelimination or fragmentation reactions. These blocking groups undergoquantitative deprotection reactions under conditions that leave thephosphorus-carboxylate moiety intact. These novel phosphinoamiditecarboxylates also enable the synthesis of new nucleoside H-phosphonitecarboxylates as well as phosphinocarboxylate-derived andphosphonocarboxylate-derived amino acids, peptides, proteins andcarbohydrates.

In one embodiment, then, phosphinoamidite carboxylates and analogsthereof are provided having the structure of formula (I)

wherein the various substituents are as follows:

R¹ is hydrogen, a protecting group removable by an elimination reaction,hydrocarbyl, substituted hydrocarbyl, heteroatom-containing hydrocarbylor substituted heteroatom-containing hydrocarbyl. Preferably, R¹ ishydrogen or an unsubstituted, substituted, heteroatom-containing orsubstituted heteroatom-containing moiety selected from the groupconsisting of alkyl, aryl, aralkyl, alkaryl, cycloalkyl,cycloalkylalkyl, cycloalkylaryl, alkenyl, cycloalkenyl, aralkenyl,alkynyl and aralkynyl. R¹ may also be a protecting group, in which caseit is removable by an elimination reaction. Protecting groups suitablefor use as R¹ will generally although not necessarily be:electron-withdrawing, -substituted aliphatic groups, particularlyelectron-withdrawing, -substituted ethyl; electron-withdrawingsubstituted phenyl; and electron-withdrawing substituted phenylethyl.Specific examples of suitable protecting groups include, by way ofexample, β-cyanoethyl, methyl-β-cyanoethyl, dimethyl-β-cyanoethyl,phenylsulfonylethyl, methyl-sulfonylethyl, p-nitrophenylsulfonylethyl,2,2,2-trichloro- 1,1-dimethylethyl, 2-(4-pyridyl)ethyl,2-(2-pyridyl)ethyl, allyl, 4-methylene-1-acetylphenol,-thiobenzoylethyl, 1,1,1,3,3,3-hexafluoro-2-propyl,2,2,2-trichloroethyl, p-nitrophenylethyl, p-cyanophenyl-ethyl,9-fluorenylmethyl, 1,3-dithionyl-2-methyl, 2-(trimethylsilyl)ethyl,2-methylthioethyl, 2-(diphenylphosphino)ethyl, 1-methyl-1-phenylethyl,3-buten-1-yl, 4-(trimethylsilyl)-2-buten-1-yl, cinnamyl,-methylcinnamyl, and 8-quinolyl.

R² and R³ are independently selected from the group consisting ofhydrocarbyl, substituted hydrocarbyl, heteroatom-containing hydrocarbyland substituted heteroatom-containing hydrocarbyl. Preferably, R² and R³are unsubstituted, substituted, heteroatom-containing or substitutedheteroatom-containing moieties selected from the group consisting ofalkyl, aryl, aralkyl, alkaryl, cycloalkyl, cycloalkylalkyl,cycloalkylaryl, alkenyl, cycloalkenyl, alkynyl and aralkynyl. That is,R² and R³ may be the same or different and are typically selected fromthe group consisting of alkyl, aryl, aralkyl, alkaryl, cycloalkyl,alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, optionally containing oneor more nonhydrocarbyl linkages such as ether linkages, thioetherlinkages, oxo linkages, amine and imine linkages, and optionallysubstituted on one or more available carbon atoms with a nonhydrocarbylsubstituent such as cyano, nitro, halo, or the like. Preferably, R² andR³ represent lower alkyl, more preferably sterically hindered loweralkyls such as isopropyl, t-butyl, isobutyl, sec-butyl, neopentyl,tert-pentyl, isopentyl, sec-pentyl, and the like. Most preferably, R²and R³ both represent isopropyl. Alternatively, R² and R³ are linked toform a mono- or polyheterocyclic ring having a total of from 1 to 3,usually 1 to 2 heteroatoms and from 1 to 3 rings. In such a case, R² andR³ together with the nitrogen atom to which they are attached represent,for example, pyrrolidino, morpholino, piperazino or piperidino. Usually,R² and R³ have a total of from 2 to 12 carbon atoms. Correspondinglypreferred NR²R³ groups include, without limitation, dimethylamino,diethylamino, diisopropylamino, dibutylamino, methylpropylamino,methylhexylamino, methylcyclohexylamino, ethylcyclopropyl-amino,ethylchloroethylamino, methylbenzylamino, methylphenylamino,thiomorpholino, methyltoluylamino, methyl-p-chlorophenylamino,methylcyclohexyl amino, bromobutylcyclohexylamino, methyl-p-cyanophenylamino, ethyl-β-cyanoethylamino, piperidino, 2,6,-dimethylpiperidino,pyrrolidino, piperazino, isopropylcyclohexylamino, and morpholino. Anexemplary NR²R³ group is diisopropylamino.

R⁴ is NR⁵R⁶, halogen or DL, wherein R⁵ and R⁶ are as defined above forR² and R³, D is O, S or NH, preferably O, and L is aheteroatom-protecting group, unsubstituted hydrocarbyl, substitutedhydrocarbyl, heteroatom-containing hydrocarbyl, or substitutedheteroatom-containing hydrocarbyl. When L is a heteroatom-protectinggroup, it is removable by an elimination reaction. Examples ofheteroatom-protecting groups that can serve as L include, but are notlimited to, those groups recited as suitable R¹ protecting groups thatare removable by an elimination reaction, i.e. β-cyanoethyl,methyl-β-cyanoethyl, dimethyl-β-cyanoethyl, phenylsulfonylethyl,methyl-sulfonylethyl, p-nitrophenyl-sulfonylethyl,2,2,2-trichloro-1,1-dimethylethyl, 2-(4-pyridyl)ethyl,2-(2-pyridyl)ethyl, allyl, 4-methylene- 1-acetylphenol,-thiobenzoylethyl, 1,1,1,3,3,3-hexafluoro-2-propyl,2,2,2-trichloroethyl, p-nitrophenylethyl, p-cyanophenyl-ethyl,9-fluorenylmethyl, 1,3-dithionyl-2-methyl, 2-(trimethylsilyl)ethyl,2-methylthioethyl, 2-(diphenylphosphino)ethyl, 1-methyl-1-phenylethyl,3-buten-1-yl, 4-(trimethylsilyl)-2-buten-1-yl, cinnamyl, -methylcinnamyland 8-quinolyl.

X is O, S, NH or NR⁷, wherein R⁷ is hydrocarbyl, substitutedhydrocarbyl, heteroatom-containing hydrocarbyl or substitutedheteroatom-containing hydrocarbyl, preferably unsubstituted,substituted, heteroatom-containing or substituted heteroatom-containingmoieties selected from the group consisting of alkyl, aryl, aralkyl,alkaryl, cycloalkyl, cycloalkylalkyl, cycloalkylaryl, alkenyl,cycloalkenyl, alkynyl and aralkynyl. Generally, however, X is O.

The subscript “n” is zero or 1, meaning that the linkage Y may or maynot be present. If present, Y is —(Y′)_(m)—(CR⁸R⁹)— wherein m is zero or1, Y′ is hydrocarbylene, substituted hydrocarbylene,heteroatom-containing hydrocarbylene, or substitutedheteroatom-containing hydrocarbylene, wherein R⁸ and R⁹ areindependently selected from the group consisting of hydrogen,hydrocarbyl, substituted hydrocarbyl, heteroatom-containing hydrocarbyland substituted heteroatom-containing hydrocarbyl, with the proviso thatwhen n is 1, m is zero and R⁸ and R⁹ are both hydrogen, then R¹ ishydrogen or a protecting group removable by an elimination reaction.Preferably, R⁸ and R⁹ are hydrogen or unsubstituted, substituted,heteroatom-containing or substituted heteroatom-containing moietiesselected from the group consisting of alkyl, aryl, aralkyl, alkaryl,cycloalkyl, cycloalkylalkyl, cycloalkylaryl, alkenyl, cycloalkenyl,alkynyl and aralkynyl. Optimally, either n is zero or n is 1 and m is 1.

Z is O, S, NH or NR¹⁰ wherein R¹⁰ is as defined for R⁷. Generally andpreferably, however, Z is O.

It should also be noted that if NR²R³ is not the same as R⁴, thecompound of formula (I) is a chiral molecule that can exist in one oftwo isomeric forms, i.e., as

These two isomers are readily separated using conventional means, e.g.,chromatographic means such as TLC or HPLC. The compounds can thus beused to prepare stereoisomerically pure modified nucleosides,oligonucleotides, and other compounds. See, for example, Wilk et al.(2000) J. Am. Chem. Soc. 122:2149-2156.

In a particularly preferred embodiment, then, the phosphinoamiditecarboxylate has the structure of formula (II)

wherein: R¹ is hydrogen, lower alkyl, or a hydroxyl-protecting groupremovable by an elimination reaction, preferably although notnecessarily an electron-withdrawing, -substituted aliphatic group; R²and R³ are lower alkyl, e.g., isopropyl, or R² and R³ are linked to forma piperidino, piperazino or morpholino ring; R⁴ is NR⁵R⁶, chloro or OLwherein R⁵ and R⁶ are as defined for R² and R³, and L is ahydroxyl-protecting group removable by an elimination reaction,generally although not necessarily selected from the group consisting ofβ-cyanoethyl, methyl-β-cyanoethyl, dimethyl-β-cyanoethyl,phenylsulfonylethyl, methylsulfonylethyl, p-nitrophenyl-sulfonylethyl,2,2,2-trichloro-1,1-dimethylethyl, 2-(4-pyridyl)ethyl,2-(2-pyridyl)ethyl, allyl, 4-methylene-1-acetylphenol,-thiobenzoylethyl, 1,1,1,3,3,3-hexafluoro-2-propyl,2,2,2-trichloroethyl, p-nitrophenylethyl, p-cyanophenyl-ethyl,9-fluorenylmethyl, 1,3-dithionyl-2-methyl, 2-(trimethylsilyl)ethyl,2-methylthioethyl, 2-(diphenylphosphino)ethyl, 1-methyl-1-phenylethyl,3-buten-1-yl, 4-(trimethylsilyl)-2-buten-1-yl, cinnamyl, -methylcinnamyland 8-quinolyl; n is zero or 1; and Y is —(Y′)_(m)—(CH₂)— wherein m iszero or 1 and Y′ is lower alkylene, with the proviso that when n is 1and m is zero, then R¹ is either hydrogen or a hydroxyl-protectinggroup.

These phosphinoamidite carboxylates and analogs thereof may be readilysynthesized from a commercially available phosphorus trihalide such asphosphorus trichloride by reaction with a secondary amine NHR²R³,wherein R² and R³ are as defined earlier. The reaction may be controlledto produce the desired mono-substituted or di-substituted reactionproduct by the stoichiometric addition of the secondary amine.Alternatively, an NR⁵R⁶ moiety or a DL moiety may be introduced alongwith an NR²R³ moiety by reaction of the phosphorus trihalide with oneequivalent of NHR²R³ and one equivalent of either HNR⁵R⁶ or H-DL,wherein R⁵, R⁶, D and L are as defined earlier. Depending on theselected reactants and the quantities used in the reaction, then, thephosphorus amide halide so produced has the structure

wherein Hal is halogen, e.g., chloro. One specific example of such areaction employs phosphorus trichloride and diisopropylamine, whichreact to produce bis-N,N-diisopropylamino chlorophosphine. The productof the reaction may be isolated and purified by vacuum distillation,recrystallization, or other techniques. The reaction of chlorophosphineswith dialkyl amines has been described and is well known to thoseskilled in the art (Schwarz et al. (1984) Tetrahedron Lett. 25:5513;Dahl et al. (1987) Nucleosides & Nucleotides 6:457; Mcbride et al.(1983) Tetrahedron Lett. 24:245). The phosphorus amide halide may thenbe converted to the phosphinoamidite carboxylate by reaction with asuitably substituted carboxylate or analog thereof, e.g.,

wherein R¹, X, Y, Z and n are as defined previously, and RG is areactive group that is displaced upon reaction with the phosphorus amidehalide.

Compounds of formula (I) are useful as phosphitylating agents, in thatthe NR²R³ and/or the R⁴ moieties are leaving groups susceptible todisplacement by nucleophilic attack, e.g., by a hydroxyl or other group.For example, the compounds can be used to phosphitylate a nucleoside oran oligonucleotide by reaction of a 3′-hydroxyl group, as follows:

In the example illustrated, A is generally H or a protected hydroxylgroup, B is a nucleobase, which may be protected with one or moreprotecting groups as known in the art, E is hydrogen, a suitable3′-hydroxyl protecting group, or a continuing oligonucleotide chain, R⁴is NR⁵R⁶, halogen or DL, and W is NR²R³, NR⁵R⁶ or DL. Typically, thereaction conditions for this phosphitylation reaction are the same asthose used in known methods of DNA synthesis, e.g., using conventionalphosphoramidite chemistry (see Beaucage and Caruthers (1981) TetrahedronLett. 22:1859-1862). It should be noted that the substituent W in thephosphitylated product is determined by R⁴ in the phosphitylatingreagent. That is, when R⁴ is halogen, e.g., chloro, W will be NR²R³.When R⁴ is DL, then W will generally be DL, unless DL and NR²R³ areselected such that DL is a better leaving group than NR²R³. When R⁴ isNR⁵R⁶, then W will be NR²R³ or NR⁵R⁶, or there may be a mixture ofreaction products wherein W is NR²R³ or NR⁵R⁶; again, the substituent inthe reaction may be controlled by appropriate selection of R², R³, R⁵and R⁶ as will be appreciated by those skilled in the art.

Generally, when the compounds of formula (I) are used to phosphitylate anucleoside or an oligonucleotide, protecting groups (e.g.,amine-protecting groups) are used to protect the nucleobase “B.”Nucleobase protecting groups and methods for protecting and deprotectingnucleobases are known in the art and described in the pertinent textsand literature, e.g.: Shaller etal. (1963) J. Am Chem Soc. 85: 3821; Tiet. al. (1982) J. Am. Chem. Soc. 104: 1316; Chaix et. al. (1989)Tetrahedron Lett. 30:71; Hagen et. al. (1989) J. Org. Chem. 54:3189;Nyilas et. al. (1988) Nucleosides & Nucleotides 7:787; Himmelsbach et.al. (1983) Tetrahedron Lett. 24: 3583; McBride et. al. (1986) J. Am.Chem. Soc. 108: 2040; and Beijer et. al. (1990) Nucl. Acids Res.18:5143.

Peptides, proteins and individual amino acids may also be phosphitylatedusing the phosphitylating agents of formula (I) at any nucleophilicsite, e.g., at the hydroxyl groups of serine, threonine and tyrosine, orat the sulfhydryl group of cysteine. It should be emphasized, however,that the phosphitylating agents of formula (I) are not limited withrespect to use in phosphitylating biomolecules such as nucleosides,oligonucleotides, peptides, proteins and amino acids, but are useful inphosphitylating any compounds having a nucleophilic site that candisplace the NR²R³ or R⁴ moieties bound to the phosphorus atom of thephosphitylating agent.

The compounds of formula (I) may also be useful as therapeutic agents,e.g., as antiviral agents or anticancer agents, and may be screened foractivity using conventional techniques. Compounds of formula (I) havingantiviral activity may be used to treat a patient afflicted with a viralinfection. Viral infections include, by way of example: retrovirusessuch as, but not limited to, HTLV-I, HTLV-II, human immunodeficiencyviruses, HTLV-III (AIDS virus), and the like; RNA viruses such as, butnot limited to, influenza type A, B, and C, mumps, measles, rhinovirus,dengue, rubella, rabies, hepatitis virus A, encephalitis virus, and thelike; and DNA viruses such as, but not limited to, herpes viruses(including herpes simplex virus-1, herpes simplex virus-2,varicella-zoster virus, Epstein-Barr virus, human cytomegalovirus, humanherpes virus 6, human herpes virus 7, and human herpes virus 8),vaccinia, papilloma virus, hepatitis virus B, and the like. Compounds offormula (I) having anticancer activity may be used to treat a patientafflicted with or susceptible to a neoplastic disease state. Neoplasticdisease states include: leukemias such as, but not limited to, acutelymphoblastic, chronic lymphocytic, acute myloblastic and chronicmylocytic leukemias; carcinomas, such as, but not limited to, those ofthe cervix, oesophagus, stomach, small intestines, colon and lungs;sarcomas, such as, but not limited to, oesteroma, osteosarcoma, lepoma,liposarcoma, hemangioma and hemangiosarcoma; melanomas, includingamelanotic and melanotic; and mixed types of neoplasias such as, but notlimited to, carcinosarcoma, lymphoid tissue type, follicular reticulum,cell sarcoma and Hodgkins Disease.

III. Nucleoside Phosphinoamidite Carboxylayes and NucleosideH-Phosphonite Carboxylates

In another embodiment of the invention, nucleoside phosphinoamiditecarboxylates, nucleoside H-phosphonite carboxylates, and analogs thereofare provided. The nucleoside phosphinoamidite carboxylates and analogsthereof have the structural formula (III)

wherein the various substituents are as follows:

A is hydrogen, hydroxyl, halogen, lower alkoxy, lower alkoxy-substitutedlower alkoxy, SH, NH₂, azide or DL wherein D is O, S or NH and L is asdefined previously, i.e., a heteroatom-protecting group, unsubstitutedhydrocarbyl, substituted hydrocarbyl, heteroatom-containing hydrocarbyl,or substituted heteroatom-containing hydrocarbyl. When A is hydroxyl,the nucleoside is a ribonucleoside, and when A is hydrogen, thenucleoside is a deoxyribonucleoside. Preferred L groups are as describedin Section II.

B is a nucleobase, generally adenine (A), thymine (T), cytosine (C),guanine (G) or uracil (U), and may be a protected form thereof, e.g.,wherein the base is protected with a protecting group such as acetyl,difluoroacetyl, trifluoroacetyl, isobutyryl or benzoyl, or B may be apurine or pyrimidine analog. Suitable analogs will be known to thoseskilled in the art and are described in the pertinent texts andliterature. Common analogs include, but are not limited to,1-methyladenine, 2-methyladenine, N⁶-methyladenine,N⁶-isopentyl-adenine, N⁶-benzoyladenine2-methylthio-N⁶-isopentyladenine, N,N-dimethyladenine, 8-bromoadenine,isocytosine, 2-thiocytosine, 3-methylcytosine, 5-methylcytosine,5-ethylcytosine, 1-acetylcytosine, 1-isobutyrylcytosine, isoguanine,1-methylguanine, 2-methylguanine, 7-methylguanine, 2,2-dimethylguanine,8-bromo-guanine, 8-chloroguanine, 8-aminoguanine, 8-methylguanine,8-thioguanine, 2-thiothymidine, 4-thiothymidine, 5-fluoro-uracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, 5-ethyluracil,5-propyluracil, 5-methoxyuracil, 5-hydroxymethyluracil,5-(carboxyhydroxymethyl)-uracil, 5-(methyl-aminomethyl)uracil,5-(carboxymethylaminomethyl)-uracil, 5-(1-propynyl)uracil, 2-thiouracil,4-thiouracil, 5-methyl-2-thiouracil, 5-(2-bromovinyl)uracil,uracil-5-oxyacetic acid, uracil-5-oxyacetic acid methyl ester,pseudouracil, 1-methylpseudouracil, queosine, inosine, 1-methylinosine,hypoxanthine, xanthine, 7-deazaxanthine, 2-aminopurine,6-hydroxyaminopurine, 6-thiopurine and 2,6-diaminopurine.

One of R¹¹ and R¹² is a blocking group and the other is

-   -   in which R¹ is hydrogen, a protecting group removable by an        elimination reaction, hydrocarbyl, substituted hydrocarbyl,        heteroatom-containing hydrocarbyl or substituted        heteroatom-containing hydrocarbyl, X and Z are as defined for        compounds of formula (I), the subscript “n” is zero or 1,        meaning that the linkage Y may or may not be present. If        present, Y is —(Y′)_(m)—(CR⁸R⁹)— wherein m is zero or 1, Y′ is        hydrocarbylene, substituted hydrocarbylene,        heteroatom-containing hydrocarbylene, or substituted        heteroatom-containing hydrocarbylene, wherein R⁸ and R⁹ are        independently selected from the group consisting of hydrogen,        hydrocarbyl, substituted hydrocarbyl, heteroatom-containing        hydrocarbyl and substituted heteroatom-containing hydrocarbyl.        Optimally, either n is zero or n is 1 and m is zero. In the        latter case, if R⁸ and R⁹ are hydrogen, then in these        phosphitylating agents R¹ is either hydrogen or a protecting        group removable by an elimination reaction. W is NR²R³, NR⁵R⁶ or        DL, wherein R², R³, R⁴, R⁵, R⁶, D and L are also as defined        previously with respect to compounds of formula (I).

Examples of blocking groups suitable as R¹¹ or R¹² include, but are notlimited to, heteratom-protecting groups removable by an eliminationreaction, generally although not necessarily selected from the groupconsisting of trityl, monomethoxytrityl (“MMT”), dimethoxytrityl(“DMT”), 9-(9-phenyl)xanthenyl (pixyl), 9-(9-p-methoxyphenyl)xanthenyl(“Mox”), acetyl, pivaloyl, 4-methoxytetrahydropyran-4-yl,tetrahydropyranyl, phenoxyacetyl, isobutyloxycarbonyl, benzyl,trialkylsilyl having from 3 to 9 carbon atoms, 9-fluorenylmethylcarbamate (“Fmoc”), 1-bis-(4-methoxyphenyl)-1′-pyrenylmethyl, and3-(imidazol-1-ylmethyl)-bis-(4′,4″-dimethoxyphenyl)methyl. Greene andWuts, Protective Groups in Organic Synthesis, supra, provides detailedinformation on the selection of suitable removable blocking groups(i.e., protecting groups) for use as R¹¹ or R¹². The blocking groups donot necessarily, however, have to be removable.

In a particularly preferred embodiment, the nucleoside phosphinoamiditecarboxylate has the structure of formula (III) as above wherein one ofR¹¹ and R¹² is a blocking group and the other is

wherein: R¹ is hydrogen, lower alkyl, or a hydroxyl-protecting groupremovable by an elimination reaction; R² and R³ are lower alkyl or arelinked to form a piperidino, piperazino or morpholino ring; n is zero or1; and Y is —(Y′)_(m)—(CH₂)— wherein m is zero or 1 and Y′ is loweralkylene.

These nucleoside phosphinoamidite carboxylates may be reacted with waterin the presence of an acid catalyst such as tetrazole, or with a weakacid, to undergo conversion to the corresponding H-phosphonites, i.e.,nucleoside compounds having the structure of formula (III) wherein oneof R¹¹ and R¹² is a blocking group, but the other has the structure offormula (VI)

wherein R¹, X, Y, Z and n are as defined above for nucleosidessubstituted with a group having the structure of formula IV.

A particularly preferred nucleoside H-phosphonite has the structure offormula (III) wherein, as above, one of R¹¹ and R¹² is a blocking group,but the other has the structure of formula (VII)

wherein: R¹ is hydrogen, lower alkyl, or a hydroxyl-protecting groupremovable by an elimination reaction; n is zero or 1; and Y is—(Y′)_(m)—(CH₂)— wherein m is zero or 1 and Y′ is lower alkylene.IV. Oligonucleotides

In another embodiment of the invention, modified oligonucleotides areprovided that contain at least one internucleotide linkage having thestructure of formula (VIII) or (X)

in which R¹, X, Y, Z and n are as defined above for the nucleosides offormula (III), wherein when the internucleotide linkage is (VIII), theoligonucleotide is a “phosphinocarboxylate” oligonucleotide or analogthereof, while when the internucleotide linkage is (X), theoligonucleotide is a “phosphonocarboxylate” oligonucleotide or analogthereof. Oligonucleotides having the phosphinocarboxylate linkage (VIII)may be generally represented as

wherein R¹, X, Y, Z, and n are as defined previously for structures(III) and (IV), A and B are as defined previously for structure (III),and R¹³ and R¹⁴ are independently hydrogen, a phosphoryl group, ablocking group, or a linkage to a sold support. Thesephosphinocarboxylate oligonucleotides are generally synthesized on asolid support. For 3′-to-5′ synthesis, a support-bound nucleosidemonomer is provided having the structure (XII)

wherein A and B are as defined above, and SS represents a solid supportor a support-bound oligonucleotide chain. Suitable solid supports aretypically polymeric, and may have a variety of forms and compositionsand derive from naturally occurring materials, naturally occurringmaterials that have been synthetically modified, or synthetic materials.The supports may be comprised of organic polymers, inorganic polymers,metals, metal oxides, or combinations thereof. Suitable substratematerials include, but are not limited to, supports that are typicallyused for solid phase chemical synthesis, such as: polymeric materials(e.g., polystyrene, polyvinyl acetate, polyvinyl alcohol, polyvinylchloride, polyvinyl pyrrolidone, polyacrylonitrile, polyacrylamide,polymethyl methacrylate, polytetrafluoroethylene, polyethylene,polypropylene, polyvinylidene fluoride, polycarbonate,polytetrafluoroethylene, divinylbenzene styrene-based polymers, andcopolymers of hydroxyethyl methacrylate and methyl methacrylate),including grafted polymers and soluble polymers; cellulosic polymers andother polysaccharides, including agarose (e.g., Sepharose®) and dextran(e.g., Sephadex®); silica and silica-based materials (e.g., silica,quartz, aluminosilicates and borosilicates); glasses (particularlycontrolled pore glass, or “CPG”) and functionalized glasses; ceramics,including metal oxides; metals; and such substrates treated with surfacecoatings, e.g., with microporous polymers (particularly cellulosicpolymers such as nitrocellulose).

The initial monomer will typically be covalently attached to the supportsurface, typically although not necessarily through a linking group asis known in the art. A linking group, if present, should have a lengthsufficient to allow a complementary oligonucleotide to bind to thecomplete support-bound oligonucleotide. The linking group may contain acleavable site to allow release of the completed oligonucleotide fromthe substrate surface after use, i.e., after completion of ahybridization assay. Cleavable sites may be restriction sites (i.e.,sites cleavable by restriction endonucleases), or they may be chemicallyor photolytically cleavable sites, as will be appreciated by those ofordinary skill in the art.

The monomer to be added has the structure of formula (XIII)

wherein R¹⁵ is a hydroxyl-protecting group and the remainingsubstituents are as defined above. Examples of methods and reagents forprotection of 5′-hydroxyl groups are well known and described in anumber of references, including Happ et. al. (1988) Nucleosides &Nucleotides 7:813; Chattopadhyaya et. al., (1980) Nucl. Acids Res.8:2039]; Seliger et. al. (1985) Nucleosides Nucleotides 4:153; Ma et.al. (1987) Nucleosides & Nucleotides 6:491; Pfleiderer et. al. (1986)Chem. Scr. 26:147; McGall et. al. (1997) J. Am. Chem. Soc. 119:5081; andScaringe et. al. (1998) J. Am. Chem. Soc. 120:11820. The couplingreaction is conducted under standard conditions used for the synthesisof oligonucleotides and conventionally employed with automatedoligonucleotide synthesizers. Such methodology will be known to thoseskilled in the art and is described in the pertinent texts andliterature, e.g., in D. M. Matteuci et al. (1980) Tetrahedron Lett.521:719 and U.S. Pat. No. 4,500,707. The product of the couplingreaction has the structure (XIV)

Following coupling, unreacted hydroxyl groups are optionally capped witha suitable capping agent. Then, if desired, the phosphinocarboxylatelinkage can be oxidized with a suitable oxidizing agent to provide thecorresponding phosphonocarboxylate linkage, shown in the followingstructure:

Next, the protecting group R¹⁵ is removed, and an additional5′-protected phosphinocarboxylate monomer is added in a similar manner.The process is repeated until the oligonucleotide is of the desiredlength. Following synthesis, the oligonucleotide may, if desired, becleaved from the solid support.

The method of the invention also lends itself to synthesis in the5′-to-3′ direction. In such a case, the initial step of the syntheticprocess involves attachment of a nucleoside monomer to a solid supportat the 5′ position, leaving the 3′ position available for covalentbinding of a subsequent monomer. In this embodiment, i.e., for 5′-to-3′synthesis, a support-bound nucleoside monomer is provided having thestructure (XV)

wherein A, B, and SS are as defined above for structure (XII). Theprotected monomer to be added has the structure of formula (XVI)

giving rise to the coupled product (XVII)

Oxidation of the phosphinocarboxylate to the phosphonocarboxylate,deprotection and successive addition of additional nucleosides may becarried out as above, followed by cleavage of the final oligonucleotideproduct from the solid support, if desired. With respect to R¹⁵,exemplary methods and reagents for orthogonal protection of 3′-hydroxylgroups are given by Koga et. al. (1991) J. Org. Chem. 56:3757 andPirrung et. al., U.S. Pat. No. 5,908,926, issued Jun. 1, 1999.

These phosphinocarboxylate and phosphonocarboxylate oligonucleotidesfind applications in a variety of areas, for example: as therapeuticagents, particularly as therapeutic agents in antisense-, ribozyme- andaptamer-based strategies; as diagnostic agents in target validation, totest selected proteins for suitability as a therapeutic target; ininvestigating the mechanism and stereochemistry of biochemicalreactions; and in the mapping of nucleic acid protein interactions.Furthermore, since the novel oligonucleotides have been found to directthe hydrolysis of complementary RNA in the presence of RNaseH, they arealso useful as agents for eliciting RNaseH activity. Finally, becausethe oligonucleotides have reduced charge relative to relative tooligonucleotides with standard phosphonate linkages, they readily passthrough body membranes and in addition have enhanced utility in the areaof mass spectrometry.

It may also be desirable to prepare substrate-bound arrays ofphosphinocarboxylate and phosphonocarboxylate oligonucleotides asprovided herein. High density oligonucleotide arrays are now well knownand in commercial use for a number of purposes. For example, theseso-called “DNA chips” or “gene chips” can be used in gene expressionanalysis and mutation detection, polymorphism analysis, mapping,evolutionary studies, and other applications. The term “array” as usedherein refers to a regular, ordered, two-dimensional arrangement ofoligonucleotides covalently bound or otherwise attached to a substratesurface. Substrate-bound arrays comprised of one or morephosphinocarboxylate or phosphonocarboxylate oligonucleotides of theinvention may be prepared using now-conventional techniques, e.g., by“spotting” pre-synthesized oligonucleotides onto designated sites of asubstrate surface, or by synthesizing the oligonucleotides in situ, on asolid support. Specific methods for forming and using oligonucleotidearrays will be known to those skilled in the art and/or are described inthe pertinent texts and literature.

It is to be understood that while the invention has been described inconjunction with the preferred specific embodiments thereof, that thedescription above as well as the example which follows are intended toillustrate and not limit the scope of the invention. Other aspects,advantages and modifications within the scope of the invention will beapparent to those skilled in the art to which the invention pertains.

All patents, patent applications, journal articles and other referencesmentioned herein are incorporated by reference in their entireties.

EXPERIMENTAL

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of synthetic organic chemistry,biochemistry, molecular biology, and the like, which are within theskill of the art. Such techniques are explained fully in the literature.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how toprepare and use the compounds disclosed and claimed herein. Efforts havebeen made to ensure accuracy with respect to numbers (e.g., amounts,temperature, etc.) but some errors and deviations should be accountedfor. Unless indicated otherwise, parts are parts by weight, temperatureis in ° C. and pressure is at or near atmospheric. Unless otherwiseindicated, all starting materials and reagents were obtainedcommercially and used without further purification.

Also, in these examples and throughout this specification and figures,the abbreviations employed have their generally accepted meanings, asfollows:

-   -   ACN acetonitrile    -   CPG control pore glass    -   CSO (1S)-(+)(10-camphorsufonyl)-oxaziridine    -   DBU 1,8-diazabicyclo[5.4.0]undec-7-ene    -   DMAP 4-dimethylaminopyridine    -   DMT dimethoxytrityl    -   DTT dithiothreitol    -   EDTA ethylenediaminetetraacetic acid    -   HEPES 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid    -   iPr isopropyl    -   ODN oligodeoxyribonucleotide    -   Py pyridine    -   TCA trichloroacetic acid    -   THF tetrahydrofuran    -   SVP snake venom phosphodiesterase

EXAMPLE 1 Preparation of bis-(N,N-diisopropylamino) chlorophosphine

A 5-liter, 3-neck, round-bottom flask was equipped with a Fredrich'scondenser, a ground glass stirrer bearing, a silicon rubber septum, andplaced under dry argon. Two liters (1,600 grams, 15.9 mmols, 7.0 eq) ofanhydrous diisopropyl amine was added to the flask. The diisopropylamine was diluted by the addition of two liters of anhydrousacetonitrile. The solution was mixed with a mechanical stirrer attachedto a glass stir rod and Teflon blade. An ice/water bath was placed underthe 5-liter flask, and the amine solution allowed to cool for 30 min.Phosphorus trichloride (313 grams, 2.3 mmols, 1.0 eq) was dissolved in 1liter of anhydrous acetonitrile in a separate dry 1-liter flask. Themechanical stirrer was set to vigorous stirring and the phosphorustrichloride solution added slowly to the stirring flask by cannula. Oncethe addition was complete, the ice/water bath was removed, and thereaction was allowed to warm to room temperature. The reaction wasstirred overnight and the extent of the reaction determined by ³¹P NMRof an aliquot of the reaction mixture using an external lock. Completeconversion of the starting material, phosphorus trichloride (δ 201 ppm),into the product at δ 134 ppm, demonstrated completion of the desiredreaction. The reaction mixture was filtered to remove the bulk of thediisopropylamine hydrochloride, which had precipitated. The precipitatewas washed with anhydrous ether and the filtrates evaporated into a2-liter round-bottom flask. The resulting evaporated product was asemi-crystalline solid, which was suspended in 1 liter of anhydroushexanes. The flask was heated on a mantel, to allow the hexanes to boil.The hot liquid was filtered through a Schlenk filter-funnel to removeresidual amine hydrochloride. This resulting clear yellow liquid wasevaporated to one half the original volume and placed in a freezer toallow the product to recrystallize. The recrystallized product wasisolated by filtration and dried in a vacuum desiccator, yielding 447grams (74% yield).

EXAMPLE 2 Synthesis of N,N-diisopropylamino dichlorophosphine

Bis-(N,N-Diisopropylamino) chlorophosphine, 10 grams (37 mmols), wasdissolved in 500 ml of anhydrous acetonitrile. Phosphorus trichloride,5.16 grams (37 mmols), was added to the solution and the reaction wasallowed to stir over-night at room temperature. The reaction was assayedfor completion by ³¹P NMR of an aliquot of the reaction using anexternal lock. Complete conversion of the starting material at δ 134 ppmto the product at δ 165 ppm was observed. The solvent was evaporatedfrom the reaction mixture on a rotary evaporator and the resulting oildistilled under vacuum yielding 5.7 grams of product (81% yield).

EXAMPLE 3 Synthesis of Acetic acid. (bis-N,N-(diethylamino)phosphino)-methyl ester

A 500 ml 3-neck round bottom flask was equipped with a reflux condenser,magnetic stir bar and two addition funnels. 13.1 grams, 200 mmol ofgranular zinc was placed in the bottom of the flask.Bis-(N,N-diethylamino)chlorophosphine (21.1 grams, 100 mmol) and methylbromoacetate (14.1 grams, 100 mmol) were each dissolved in 200 ml ofanhydrous ether. The two ether solutions were placed in the droppingfunnels and the reflux condenser fitted with a dry argon line. 30 ml ofeach solution was ran into the round bottom flask and the mixturestirred vigorously. The reaction mixture was heated with a heat gununtil the ether boiled. The heat was kept on the mixture until thereaction mixture became clear and slightly yellow. At this point, thereaction continued to boil without addition of heat due to theexothermic nature of the reaction. Steady addition of the two ethersolutions kept the reaction at a vigorous boil. After the addition wascomplete, the reaction was kept at a boil by the addition of heat for 10min. The reaction was allowed to cool and an aliquot was removed for ³¹PNMR. The ³¹P NMR demonstrated complete conversion of the startingmaterial δ 159.2 ppm to the product at δ 50.9 ppm. The reaction mixturewas decanted leaving behind the unreacted zinc and the ether was removedunder vacuum on a rotary evaporator. The product was isolated bytriturating with pentanes. The product was characterized by ¹H NMRsinglet δ 3.66 (integration 3), doublet δ 2.93, 2.91 (integration 2),quartet δ 2.72 (integration 8), triplet δ 1.26 (integration 12).Electron Impact Mass Spectrometry gave a molecular radical of 248 m/ewith fragmentation loss of CH₂COOCH₃ at 175 m/e. This correctlyidentified product gave different ³¹P NMR characterization than theproduct reported by Novikova et. al. (1976), Zhurnal Obshchei Khimii46:575. However, upon attempted distillation, the acetic acid(bis-N,N-(diethylamino) phosphino)-methyl ester decomposed into acomplex reaction mixture. The decomposed, unidentified, productscontained a³¹P NMR peak at the misreported chemical shift of δ 82 ppm(−82 ppm).

EXAMPLE 4 Synthesis of Acetic acid, (bis-N,N-(diisopropylamino)phosphino)-methyl ester

In a 1000 ml three neck round bottom flask, granular zinc metal 7.2grams, 110 mmol was placed in the bottom with a magnetic stir-bar. Thethree neck flask was fitted with a Fredrich's condenser and two 500 mladdition funnels. The addition funnels were filled with minimum-volumeether solutions of bis-N,N-diisopropylaminochlorophosphine (20 grams, 75mmol, 1.0 eq), and methylbromoacetate (11.6 grams, 82.5 mmol, 1.1 eq).Approximately one third of each solution was added to the flask and themixture was heated with a heat gun until the Reformatsky reaction wasinitiated. Once initiated, the reaction was continued by constantaddition of the two solutions. Once the addition was complete, thereaction was stirred for 15 min. The extent of the reaction wasdetermined by ³¹P NMR. The starting material gave a chemical shift of δ135 ppm and the product a chemical shift of δ 49 ppm. The reaction wasfitted with a mantel and the ether was refluxed until the startingmaterial was consumed as monitored by ³¹P NMR. The reaction wasevaporated and the resulting oil was extracted with anhydrous hexanes.The hexanes fraction was evaporated and the product distilled undervacuum to give a colorless oil, 11.8 grams, 54% yield. The product wascharacterized by ¹H NMR singlet δ 3.68 (integration 3), multiplet δ 3.55(integration 4), doublet δ 2.93, 2.91 (integration 2), multiplet δ 1.30(integration 24). Electron Impact Mass Spectrometry gave a molecularradical of 304 m/e with fragmentation loss of CH₂COOCH₃ at 231 m/e.

EXAMPLE 5 Synthesis of Acetic acid (bis-N,N-(diisopropylamino)phosphino)-dimethylcyano-ethyl ester

Dimethylcyanoethylbromoacetate was synthesized from bromoacetyl bromideand 3-hydroxy-3methylbutyronitrile. The bromoacetyl bromide (108 grams,600 mmol) was dissolved in 500 ml of anhydrous toluene in a 1 literround bottom flask. The 3-hydroxy-3methylbutrionitrile (50 grams, 500mmol) was added slowly with stirring. The round bottom flask was fittedwith a Friedrich's condenser and a drying tube. The outlet on the dryingtube was fitted with a thick hose and the exhaust taken to an acid trap.The reaction was heated to reflux using a mantel and the reaction wasrefluxed to effluvium HBr. The reaction was refluxed overnight thencooled to room temperature and evaporated to an oil on a rotaryevaporator. The oil was distilled under vacuum. The first fraction wasof wide boiling range and was discarded. The second, major, fraction wasof constant boiling range and gave 97.3 grams of a, clear, colorlessliquid. ¹H NMR of this material gave three singlets: δ 3.75 (relativeintegration 2), δ 2.86 (relative integration 2), δ 1.53 (relativeintegration 6). Bis-N,N-diisopropylamino-chlorophosphine (20 grams, 75mmol, 1.0 eq) was dissolved in 160 ml of anhydrous THF in a 500 ml roundbottom flask. The flask was stoppered and allowed to stir until all thephosphine was dissolved. Once dissolved, 100 ml of anhydrous ether wasadded to the phosphine solution. Dimethylcyanoethylbromoacetate (MW220), 18.2 grams, 82.5 mmol, 1.1 eq was dissolved in 75 ml of anhydrousether. Granular zinc metal, 7.2 grams, 110 mmol was placed in the bottomof a three neck round bottom flask along with a magnetic stir-bar. Theround bottom flask was fitted with a Friedrich's condenser and twoaddition funnels. The phosphine solution was placed in one of theaddition funnels and the bromoacetate was placed in the other. 85 ml ofthe phosphine solution and 25 ml of the bromoacetate solution were addedto the zinc metal. The reaction was heated with a heat gun until theether vigorously refluxed. The heat was removed and the reflux allowedto quite. Heat was again applied and the process was repeated until anexothermic reaction was noticeable. The slightly cloudy, colorlessreaction became clear and slightly yellow once the exothermic reactionbegan. The reaction was continued by addition of the two solutions andthe reaction was kept at reflux by use of the heat gun. The reaction waskept at reflux for 30 min and then allowed to cool. The reaction wasmonitored for completeness by ³¹P NMR. The starting material at δ 135ppm was converted to a single product at δ 48 ppm. The cooled reactionmixture was transferred to a 1-liter round bottom flask and the THF andether removed on a rotary evaporator. The resulting viscous oil wasextracted three times with anhydrous hexanes. The extraction convertedthe viscous oil to a solid and the solid was dissolved in anhydrousacetonitrile. The acetonitrile solution was extracted twice withanhydrous hexanes and all the hexanes fractions combined and evaporatedunder vacuum to a slightly yellow oil. The acetonitrile solution wasanalyzed by ³¹P NMR for absence of the product at δ 48 ppm, anddiscarded. The isolated product was further purified by dissolving inanhydrous hexanes and placing in a freezer overnight. The resultinghexanes solution was decanted into a clean, dry, 500 ml round bottomflask. The hexanes were removed by evaporation to give 16.4 grams ofpurified product, 88% yield. The product was characterized by ¹H NMR:multiplet δ 3.48 (integration 4), singlet δ 2.99 (integration 2),doublet δ 2.80, 2.78 (integration 2), multiplet δ 1.30 (integration 24).Electron Impact Mass Spectrometry gave a molecular radical of 371 m/ewith fragmentation loss of CH₂COOC(CH₃)₂CH₂CN at 231 m/e.

EXAMPLE 6 Synthesis of Formic acid (bis-N,N-(diisopropylamino)phosphino)-methyl ester

Methyl chloroformate (79.7 grams, 843 mmol, 3.0 eq) was dissolved in 250ml of anhydrous tetrahydrofuran (THF) in a dry 500 ml round bottom flaskcontaining activated 3A molecular sieves and stored overnight to removetraces of HCl. Bis-N,N-diisopropylaminochlorophosphine (75.0 grams, 281mmol, 1.0 eq) was dissolved in 200 ml anhydrous THF in a dry 250 mlround bottom flask. In a separate 500 ml round bottom flask, 200 ml ofanhydrous THF was added with a magnetic stir bar. Lithium aluminumhydride (LiAlH₄, 10.7 grams, 281 mmol, 1.0 eq) was added slowly withstirring. The LiAlH₄ solution was fitted with an argon line and cooledin an ice/water bath. The phosphine solution was slowly cannulated intothe stirring LiAlH₄ solution over 10 min. After addition was complete,the ice/water bath was removed and the reduced phosphine solution wasallowed to warm to room temperature. The reaction was checked by ³¹PNMR. The reaction was complete when the starting material (singlet,135.2 ppm) was converted completely to the product (singlet, 38.8 ppm).The reaction mixture was filtered through a sintered glass Schlenkfunnel into a dry 500 ml round bottom flask containing a magneticstir-bar and 20 grams of sodium metal spheres under argon. The roundbottom flask was fitted with a Friedrich's condenser and the solutionrefluxed under argon for 3 hours using a mantel. The mantel was removedand the resulting solution allowed to cool to room temperature. Themethyl chloroformate solution was transferred by cannula to a dry 1000ml round bottom flask containing a magnetic stir-bar. The methylchloroformate solution was fitted with an argon line and cooled in anice/water bath. The phosphine solution was cannulated into the stirringsolution of the methyl chloroformate. After the addition was complete,the reaction was monitored by ³¹P NMR. The reaction was complete whenthe starting material (singlet, 43.2 ppm) was converted to the product(singlet, 52.0 ppm) as determined by ³¹P NMR. The reaction was quenchedby the dropwise addition of triethyl amine, 85.2 grams, 843 mmol, 3.0eq. The entire mixture was evaporated on a rotary evaporator and theresidual oil triturated several times with anhydrous hexanes. Thehexanes were evaporated and resulting oil distilled under high vacuum.The product gave a clear, colorless, liquid which distilled at 95° C. at0.04 mm Hg. The purified product was dissolved in deuteratedacetonitrile and characterized by ¹H NMR; singlet δ 3.69 (integration3), multiplet δ 3.55 (integration 4), multiplet δ 1.30 (integration 24).Electron Impact Mass Spectrometry gave a molecular radical of 290 m/ewith fragmentation loss of COOCH₃ at 231 m/e.

EXAMPLE 7 Synthesis of Formic acid, (bis-N,N-(diisopropylamino)phosphino)-cyanoethyl ester

Cyanoethyl chloroformate was synthesized from phosgene and3-hydroxyproprionitrile. The phosgene was obtained as a 20% solution intoluene. The toluene solution (500 ml, 100 grams, 1.0 mol) was placed ina 1-liter round bottom flask with a magnetic stir-bar. The3-hydroxyproprionitrile (23.4 grams, 0.33 mol) was dissolved inanhydrous tetrahydrofuran (THF) and placed in an addition funnel. TheTHF solution was added dropwise to the phosgene solution and thereaction mixture allowed to stir at room temperature overnight. Thereaction mixture was evaporated to an oil using a rotary evaporatorplaced in a fume hood. The vacuum was applied using a Teflon-headdiaphragm pump and the exhaust bubbled through a sodium bicarbonatesolution to trap and neutralize the excess phosgene. The toluene removedfrom the reaction was poured into methanol and further neutralized withwater before disposal. The resulting product was isolated and purifiedby distillation under vacuum. The ¹H NMR of the distilled material inCDCl₃ gave 2 triplets centered at δ 2.77 ppm (relative integration 2.0),and δ 4.44 ppm (relative integration 2.0). The ¹³C NMR of the distilledmaterial in CDCl₃ gave 4 decoupled singlets at δ 148.95, δ 114.62, δ63.94, and δ 16.18. In a 500 ml round bottom flask 200 ml of anhydrousTHF was added with a magnetic stir bar. Lithium aluminum hydride (10.7grams, 281 mmol, 1.0 eq) was added slowly with stirring. The LiAlH₄solution was fitted with an argon line and cooled in an ice/water bath.Bis-N,N-diisopropylaminochlorophosphine (75.0 grams, 281 mmol, 1.0 eq)was dissolved in a minimum volume of anhydrous THF. The phosphinesolution was slowly cannulated into the stirring LiAlH₄ solution over 10min. After addition was complete, the ice/water bath was removed and thereduced phosphine solution was allowed to warm to room temperature. Thereaction was checked by ³¹P NMR. The reaction was complete when thestarting material (singlet, δ 135.2 ppm) was converted completely to theproduct (singlet, δ 42.8 ppm). The reaction mixture was filtered througha sintered glass Schlenk funnel into a dry 500 ml round bottom flaskcontaining a magnetic stir-bar and 20 grams of sodium metal spheresunder argon. The round bottom flask was fitted with a Friedrich'scondenser and the solution refluxed under argon for 3 hours using amantel. The mantel was removed and the resulting solution allowed tocool to room temperature. The cyanoethylchloroformate (843 mmol, 112.5grams) was dissolved in anhydrous THF, 600 ml in a 2-liter flask, andcooled in an ice/water bath. The reduced phosphine was added to thechloroformate by cannula and allowed to stir for one hour. The ice bathwas removed and the reaction was allowed to warm to room temperature.The reaction was checked for completion by ³¹P NMR. The product gave asingle peak at δ 52.0 ppm. The reaction was continued until the startingmaterial was completely converted to the product. The reaction mixturewas then cannulated into separate, dry, 2-liter round bottom flaskcontaining 500 ml of anhydrous triethylamine. The resulting precipitatewas removed by filtration. The filtrate was evaporated to a thick oiland the oil was extracted with anhydrous hexanes. The hexanes wereevaporated and the product purified by vacuum distillation. The productwas obtained as a colorless oil (65.6 grams), 71% yield. The purifiedproduct gave a ³¹P NMR peak at δ 53.0 ppm. The purified product wasdissolved in deuterated acetonitrile and characterized by ¹H NMR;doublet δ 1.34, 1.30 (integration 24), triplet δ 2.86 (integration 2),multiplet δ 3.52 (integration 4), triplet δ 4.37 (integration 2).Electron Impact Mass Spectrometry gave a molecular radical of 329 m/ewith fragmentation with loss of COOCH₂CH₂CN at 231 m/e.

EXAMPLE 8 Synthesis of Acetic acid,(N,N-(diisopropylamino)cyanoethoxyphosphino) -dimethylcyanoethyl ester

Acetic acid, (bis-N,N-(diisopropylamino) phosphino)-dimethylcyanoethylester, 5 grams, 13.5 mmol, was dissolved in 150 ml of anhydrousdichloromethane in a 250 ml round bottom flask. 3-Hydroxyproprionitrile,1 gram, 14.8 mmol, was added to the phosphine mixture and the solutionstirred using a magnetic stir-bar. Tetrazole (0.75 grams, 10.8 mmol) wasadded to the mixture and the reaction allowed to stir overnight. Themixture was assayed for completion by TLC in 50/50, hexanes/ethylacetate. The product was isolated by silica gel, flash chromatographyusing an ethyl acetate gradient in hexanes. The product was evaporatedto an oil yielding 4.3 grams, 12.6 mmol (94% yield) and analyzed by ³¹PNMR δ 124.1 ppm, and EI Mass spectrometry giving an exact mass of 341.19(341.39 calculated).

EXAMPLE 9 General Method for Synthesis of Protected NucleosideCarboxylic Acid Phosphinoamidites

The synthesis of protected nucleoside carboxylic acid phosphinoamiditeswas accomplished by the general procedure as follows. The protectednucleosides were dissolved in anhydrous dichloromethane at aconcentration of 0.05 to 0.1 M depending upon their solubility. Thecorresponding carboxylic acid bis-aminophosphonite ester, 1.2 molarequivalents, was added to the dichloromethane solution with stirring.After complete dissolution of the phosphonite, 0.8 molar equivalents oftetrazole was added to the reaction mixture. The tetrazole was added bypipette from a 0.45 M solution of tetrazole in anhydrous acetonitrile.The reaction was allowed to stir for 24 hr. and was then analyzed forextent of reaction by ³¹P NMR and silica gel TLC (eluted with ethylacetate). The reaction was determined to be complete by spot-to-spotconversion of the protected nucleoside starting material to fastereluting spots on TLC or by loss of the bis-aminophosphonite esterstarting material by ³¹P NMR. The reaction was quenched by addition ofan equal volume of concentrated sodium bicarbonate. The mixture wasstirred to produce an emulsion then transferred to a separatory funnel.The emulsion was allowed to separate, and the dichloromethane layerremoved to an Erlenmeyer flask containing anhydrous sodium sulfate. Thesolution was allowed to dry over the sodium sulfate for 1 hour thendecanted to a round bottom flask. The sodium sulfate was rinsed withanhydrous dichloromethane and the rinsing added to the round bottomflask. The dichloromethane was removed by evaporation on a rotaryevaporator and the resulting viscous oil purified by columnchromatography on silica gel. The column was equilibrated withhexanes/ethyl acetate (50/50, vol./vol.). The viscous oil wasredissolved in a minimum volume of ethyl acetate, which was then dilutedwith hexanes up to a 50/50 solution, if allowed by solubility. Thehexanes/ethyl acetate solution was added to the top of the column andthe products were eluted with a step-gradient of 50/50 hexanes/ethylacetate (vol./vol) to 100% ethyl acetate. Fractions containing UV-activematerial were collected and analyzed by silica gel TLC (eluted withethyl acetate). Those fractions, which contained the product werecollated and evaporated to a foam on a rotary evaporator. The resultingfoam product was dried overnight in vacuo and analyzed by ³¹P NMR, andFAB mass spectroscopy.

EXAMPLE 10 Synthesis of 3′-O-(diisopropylamino)-phosphinoformic acidmethyl ester -5′-O-di-p-anisylphenylmethyl thymidine

5′-DMT thymidine (10 grams, 18.4 mmoles) was reacted with formic acid,(bis(diisopropylamino) phosphino)-methyl ester (5.4 grams, 18.4 moles)in the presence of tetrazole (1.03 grams, 14.7 mmoles) for 48 hrs. Theproduct was purified on silica gel by eluting the column with 50/50hexanes/ethyl acetate giving 9.17 grams (68% yield). Fractions from thecolumn were collected and evaporated to a foam. The resulting purifiedphosphinoamidite was analyzed by ³¹P NMR, giving a set of diastereomersat δ 107.43 and δ 108.23 ppm, and FAB Mass Spectroscopy FAB+734 m/e(m+1), FAB−732 m/e (m−1).

EXAMPLE 11 Synthesis of 3′-O-(diisopropylamino)-phosphinoformic acidβ-cyanoethyl ester-5-O-di-p-anisylphenylmethyl thymidine

5′-DMT thymidine (10 grams, 18.4 mmoles) was reacted with formic acid,(bis (diisopropylamino) phosphino)-cyanoethyl ester (6.1 grams, 18.4mmoles), in the presence of tetrazole (1.03 grams, 14.7 mmoles) for 48hrs. The product was purified on silica gel by eluting the column with50/50 hexanes/ethyl acetate and the resulting purified product gave 9.97grams (74% yield). Fractions from the column were collected andevaporated to a foam. The resulting purified phosphinoamidite wasanalyzed by ³¹P NMR, giving a set of diastereomers at δ 108.38 and δ108.42 ppm, and FAB Mass Spectroscopy FAB+733 m/e (m+1), FAB−731 m/e(m−1).

EXAMPLE 12 Synthesis of 3′-O-(diisopropylamino)-phosphinoformic acidmethyl ester-5′-O-di-p-anisylphenylmethyl-N-4-benzoylcytosine

5′-DMT-N-4-benzoylcytosine (10 grams, 15.8 mmoles) was reacted withformic acid, (bis(diisopropylamino) phosphino)-methyl ester (4.6 grams,15.8 mmoles), in the presence of tetrazole (0.89 grams, 12.6 mmoles) for48 hrs. The product was purified on silica gel by eluting the columnwith 75/25 hexanes/ethyl acetate and the resulting purified product gave8.96 grams (69% yield). Fractions from the column were collected andevaporated to a foam. The resulting purified phosphinoamidite wasanalyzed by ³¹P NMR, giving a set of diastereomers at δ 107.75 and δ109.32 ppm, and FAB Mass Spectroscopy FAB+823 m/e (m+1), FAB−821 m/e(m−1).

EXAMPLE 13 Synthesis of 3′-O-(diisopropylamino)-phosphinoformic acidβ-cyanoethyl ester-5′-O-di-p-anisylphenylmethyl-N-4-benzoylcytosine

5′-DMT-N-4-benzoylcytosine (10 grams, 15.8 mmoles) was reacted withformic acid, (bis(diisopropylamino) phosphino)-cyanoethyl ester (5.2grams, 15.8 mmoles), in the presence of tetrazole (0.89 grams, 12.6mmoles) for 48 hrs. The product was purified on silica gel by elutingthe column with 75/25 hexanes/ethyl acetate, and the resulting purifiedproduct gave 9.93 grams (73% yield). Fractions from the column werecollected and evaporated to a foam. The resulting purifiedphosphinoamidite was analyzed by ³¹P NMR, giving a set of diastereomersat δ 107.66 and δ 109.22 ppm, and FAB Mass Spectroscopy FAB+862 m/e(m+1), FAB−860 m/e (m−1).

EXAMPLE 14 Synthesis of 3′-O-(diisopropylamino)-phosphinoformic acidmethyl ester-5′-O-di-p-anisylphenylmethyl-N-4-acetylcytosine

5′-DMT-N-4-acetylcytosine (10 grams, 17.5 mmoles) was reacted withformic acid, (bis (diisopropylamino) phosphino)-methyl ester (5.1 grams,17.5 mmoles), in the presence of tetrazole (0.98 grams, 14.0 mmoles) for24 hrs. The product was purified on silica gel by eluting the columnwith 100% ethyl acetate, and the resulting purified product gave 10.24grams (77% yield). Fractions from the column were collected andevaporated to a foam. The resulting purified phosphinoamidite wasanalyzed by ³¹P NMR, giving a set of diastereomers at δ 107.91 and δ109.48 ppm, and FAB Mass Spectroscopy FAB+761 m/e (m+1), FAB−759 m/e(m−1).

EXAMPLE 15 Synthesis of 3′-O-(diisopropylamino)-phosphinoformic acidβ-cyanoethyl ester-5′-O-di-p-anisylphenylmethyl-N-4-acetylcytosine

5′-DMT-N-4-acetylcytosine (10 grams, 17.5 mmoles) was reacted withformic acid, (bis (diisopropylamino) phosphino)-cyanoethyl ester (5.8grams, 17.5 mmoles), in the presence of tetrazole (0.98 grams, 14.0mmoles) for 48 hrs. The product was purified on silica gel by elutingthe column with 100% ethyl acetate, and the resulting purified productgave 11.33 grams (81% yield). Fractions from the column were collectedand evaporated to a foam. The resulting purified phosphinoamidite wasanalyzed by ³¹P NMR, giving a set of diastereomers at δ 108.58 and δ109.50 ppm, and FAB Mass Spectroscopy FAB+800 m/e (m+1), FAB−798 m/e(m−1).

EXAMPLE 16 Synthesis of 3′-O-(diisopropylamino)-phosphinoformic acidmethyl ester-5′-O-di-p-anisylphenylmethyl-9-N-benzoyl adenosine

5′-DMT-N-9-benzoyl adenosine (10 grams, 15.3 mmoles) was reacted withformic acid, (bis (diisopropylamino) phosphino)-methyl ester (4.43grams, 15.3 mmoles), in the presence of tetrazole (0.86 grams, 12.24mmoles) for 24 hrs. The product was purified on silica gel by elutingthe column with 50/50 hexanes/ethyl acetate, and the resulting purifiedproduct gave 10.23 grams (79% yield). Fractions from the column werecollected and evaporated to a foam. The resulting purifiedphosphinoamidite was analyzed by ³¹P NMR, giving a set of diastereomersat δ 107.68 and δ 108.56 ppm, and FAB Mass Spectroscopy FAB+847 m/e(m+1), FAB−845 m/e (m−1).

EXAMPLE 17 Synthesis of 3′-O-(diisopropylamino)-phosphinoformic acidβ-cyanoethyl ester-5′-O-di-p-anisylphenylmethyl-9-N-benzoyl adenosine

5′-DMT-N-9-benzoyl adenosine (10 grams, 15.3 mmoles) was reacted withformic acid, (bis (diisopropylamino) phosphino)-cyanoethyl ester (5.03grams, 15.3 mmoles), in the presence of tetrazole (0.86 grams, 12.24mmoles) for 24 hrs. The product was purified on silica gel by elutingthe column with 50/50 hexanes/ethyl acetate, and the resulting purifiedproduct gave 8.40 grams (62% yield). Fractions from the column werecollected and evaporated to a foam. The resulting purifiedphosphinoamidite was analyzed by ³¹P NMR, giving a set of diastereomersat δ 108.56 and δ 108.67 ppm, and FAB Mass Spectroscopy FAB+886 m/e(m+1), FAB−884 m/e (m−1).

EXAMPLE 18 Synthesis of 3′-O-(diisopropylamino)-phosphinoformic acidmethyl ester-5′-O-di-p-anisylphenylmethyl-N-2-isobutyrylguanosine

5′-DMT-N-2-isobutyrylguanosine (10 grams, 15.7 mmoles) was reacted withformic acid, (bis (diisopropylamino) phosphino)-methyl ester (4.55grams, 15.7 mmoles), in the presence of tetrazole (0.88 grams, 12.56mmoles) for 24 hrs. The product was purified on silica gel by elutingthe column with 100% ethyl acetate, and the resulting purified productgave 10.66 grams (82% yield). Fractions from the column were collectedand evaporated to a foam. The resulting purified phosphinoamidite wasanalyzed by ³¹P NMR, giving a set of diastereomers at δ 107.60 and δ108.35 ppm, and FAB Mass Spectroscopy FAB+829 m/e (m+1), FAB−827 m/e(m−1).

EXAMPLE 19 Synthesis of 3′-O-(diisopropylamino)-phosphinoformic acidβ-cyanoethyl ester-5′-O-di-p-anisylphenylmethyl-N-2-isobutyrylguanosine

5′-DMT-N-2-isobutyrylguanosine (10 grams, 15.7 mmoles) was reacted withformic acid, (bis (diisopropylamino) phosphino)-cyanoethyl ester (5.17grams, 15.7 mmoles), in the presence of tetrazole (0.88 grams, 12.56mmoles) for 24 hrs. The product was purified on silica gel by elutingthe column with 100% ethyl acetate, and the resulting purified productgave 10.89 grams (80% yield). Fractions from the column were collectedand evaporated to a foam. The resulting purified phosphinoamidite wasanalyzed by ³¹P NMR, giving a set of diastereomers at δ 108.36 and δ108.45 ppm, and FAB Mass Spectroscopy FAB+868 m/e (m+1), FAB−866 m/e(m−1).

EXAMPLE 20 Synthesis of 3′-O-(diisopropylamino)-phosphinoacetic acidmethyl ester-5′-O-di-p-anisylphenylmethyl thymidine

5′-DMT thymidine (10 grams, 18.4 mmoles) was reacted with acetic acid,(bis-(diisopropylamino) phosphino)-methyl ester (5.6 grams, 18.4mmoles), in the presence of tetrazole (1.03 grams, 14.7 mmoles) for 48hrs. The product was purified on silica gel by eluting the column with50/50 hexanes/ethyl acetate, giving 11.55 grams (84% yield). Fractionsfrom the column were collected and evaporated to a foam. The resultingpurified phosphinoamidite was analyzed by ³¹P NMR, giving a set ofdiastereomers at δ 120.6 and δ 120.8 ppm, and FAB Mass SpectroscopyFAB+748 m/e (m+1), FAB−746 m/e (m−1).

EXAMPLE 21 Synthesis of 3′-O-(diisopropylamino)-phosphinoacetic aciddimethyl-β-cyanoethyl ester-5′-O-di-p-anisylphenylmethyl thymidine:

5′-DMT thymidine (10 grams, 18.4 mmoles) was reacted with acetic acid,(bis(diisopropylamino) phosphino)-dimethylcyanoethylethyl ester (6.8grams, 18.4 mmoles), in the presence of tetrazole (1.03 grams, 14.7mmoles) for 48 hrs. The product was purified on silica gel by elutingthe column with 50/50 hexanes/ethyl acetate, giving 12.88 grams (86%yield). Fractions from the column were collected and evaporated to afoam. The resulting purified phosphinoamidite was analyzed by ³¹P NMR,giving a set of diastereomers at δ 120.3 and δ 120.8 ppm, and FAB MassSpectroscopy FAB+815 m/e (m+1), FAB−813 m/e (m−1).

EXAMPLE 22 Synthesis of 3′-O-(diisopropylamino)-phosphinoacetic acidmethyl ester-5′-O-di-p-anisylphenylmethyl-N-4-benzoylcytosine

5′-DMT-N-4-benzoylcytosine (10 grams, 15.8 mmoles) was reacted withacetic acid, (bis-(diisopropylamino) phosphino)-methyl ester (4.8 grams,15.8 mmoles), in the presence of tetrazole (0.89 grams, 12.64 mmoles)for 24 hrs. The product was purified on silica gel by eluting the columnwith 50/50 hexanes/ethyl acetate, giving 8.98 grams (68% yield).Fractions from the column were collected and evaporated to a foam. Theresulting purified phosphinoamidite was analyzed by 31P NMR, giving aset of diastereomers at δ 121.1 and δ 122.0 ppm, and FAB MassSpectroscopy FAB+837 m/e (m+1), FAB−835 m/e (m−1).

EXAMPLE 23 Synthesis of 3′-O-(diisopropylamino)-phosphinoacetic aciddimethyl-β-cyanoethyl ester-5′-O-di-p-anisylphenylmethyl-N-4-benzoylcytosine

5′-DMT-N-4-benzoylcytosine (10 grams, 15.8 mmoles) was reacted withacetic acid, (bis(diisopropylamino) phosphino)-dimethylcyanoethylethylester (5.86 grams, 15.8 mmoles), in the presence of tetrazole (0.89grams, 12.64 mmoles) for 24 hrs. The product was purified on silica gelby eluting the column with 50/50 hexanes/ethyl acetate, giving 10.56grams (74% yield). Fractions from the column were collected andevaporated to a foam. The resulting purified phosphinoamidite wasanalyzed by ³¹P NMR, giving a set of diastereomers at δ 121.5 and δ121.1 ppm, and FAB Mass Spectroscopy FAB+904 m/e (m+1), FAB−902 m/e(m−1).

EXAMPLE 24 Synthesis of 3′-O-(diisopropylamino)-phosphinoacetic acidmethyl ester-5′-O-di-p-anisylphenylmethyl-N-4-acetylcytosine

5′-DMT-N-4-acetylcytosine (10 grams, 17.5 mmoles) was reacted withacetic acid, (bis-(diisopropylamino) phosphino)-methyl ester (5.3 grams,17.5 mmoles) in the presence of tetrazole (0.98 grams, 14.0 mmoles) for24 hrs. The product was purified on silica gel by eluting the columnwith 50/50 hexanes/ethyl acetate, giving 11.12 grams (76% yield).Fractions from the column were collected and evaporated to a foam. Theresulting purified phosphinoamidite was analyzed by ³¹ P NMR, giving aset of diastereomers at δ 121.1 and δ 121.5 ppm, and FAB MassSpectroscopy FAB+837 m/e (m+1), FAB−835 m/e (m−1).

EXAMPLE 25 Synthesis of 3′-O-(diisopropylamino)-phosphinoacetic aciddimethyl-β-cyanoethylester-5′-O-di-p-anisylphenylmethyl-N-4-acetylcytosine

5′-DMT-N-4-acetylcytosine (10 grams, 17.5 mmoles) was reacted withacetic acid, (bis-(diisopropylamino) phosphino)-dimethylcyanoethyl ester(6.5 grams, 17.5 mmoles), in the presence of tetrazole (0.98 grams, 14.0mmoles) for 24 hrs. The product was purified on silica gel by elutingthe column with 100% ethyl acetate, giving 11.48 grams (78% yield).Fractions from the column were collected and evaporated to a foam. Theresulting purified phosphinoamidite was analyzed by ³¹P NMR, giving aset of diastereomers at δ 121.3 and δ 121.8 ppm, and FAB MassSpectroscopy FAB+842 m/e (m+1), FAB−840 m/e (m−1).

EXAMPLE 26 Synthesis of 3′-O-(diisopropylamino)-phosphinoacetic acidmethyl ester-5′-O-di-p-anisylphenylmethyl-N-9-benzoyl adenosine

5′-DMT-N-9-benzoyl adenosine (10 grams, 15.3 mmoles) was reacted withacetic acid, (bis-(diisopropylamino) phosphino)-methyl ester (4.7 grams,15.3 mmoles), in the presence of tetrazole (0.86 grams, 12.24 mmoles)for 24 hrs. The product was purified on silica gel by eluting the columnwith 50/50 hexanes/ethyl acetate, giving 10.53 grams (80% yield).Fractions from the column were collected and evaporated to a foam. Theresulting purified phosphinoamidite was analyzed by ³¹P NMR, giving aset of diastereomers at δ 120.7 and δ 121.6 ppm, and FAB MassSpectroscopy FAB+861 m/e (m+1), FAB−859 m/e (m−1).

EXAMPLE 27 Synthesis of 3′-O-(diisopropylamino)-phosphinoacetic aciddimethyl-β-cyanoethyl ester-5′-O-di-p-anisylphenylmethyl-N-9-benzoyladenosine

5′-DMT-N-9-benzoyl adenosine (10 grams, 15.3 mmoles) was reacted withacetic acid, (bis-(diisopropylamino) phosphino)-dimethylcyanoethyl ester(5.7 grams, 15.3 mmoles), in the presence of tetrazole (0.86 grams,12.24 mmoles) for 24 hrs. The product was purified on silica gel byeluting the column with 50/50 hexanes/ethyl acetate, giving 10.64 grams(75% yield). Fractions from the column were collected and evaporated toa foam. The resulting purified phosphinoamidite was analyzed by ³¹P NMR,giving a set of diastereomers at δ 120.8 and δ 121.6 ppm, and FAB MassSpectroscopy FAB+928 m/e (m+1), FAB−926 m/e (m−1).

EXAMPLE 28 Synthesis of 3′-O-(diisopropylamino)-phosphinoacetic acidmethyl ester-5′-O-di-p-anisylphenylmethyl-N-2-isobutyrylguanosine

5′-DMT-N-2-isobutyrylguanosine (10 grams, 15.7 mmoles) was reacted withacetic acid, (bis-(diisopropylamino) phosphino)-methyl ester (4.8 grams,15.7 mmoles), in the presence of tetrazole (0.88 grams, 12.56 mmoles)for 24 hrs. The product was purified on silica gel by eluting the columnwith 100% ethyl acetate, giving 10.97 grams (83% yield). Fractions fromthe column were collected and evaporated to a foam. The resultingpurified phosphinoamidite was analyzed by ³¹P NMR, giving a set ofdiastereomers at δ 121.3 and δ 121.7 ppm, and FAB Mass SpectroscopyFAB+843 m/e (m+1), FAB−841 m/e (m−1).

EXAMPLE 29 Synthesis of 3′-O-(diisopropylamino)-phosphinoacetic aciddimethyl-β-cyanoethylester-5′-O-di-p-anisylphenylmethyl-N-2-isobutyrylguanosine

5′-DMT-N-2-isobutyrylguanosine (10 grams, 15.7 mmoles) was reacted withacetic acid, (bis-(diisopropylamino) phosphino)-dimethylcyanoethyl ester(5.8 grams, 15.7 mmoles), in the presence of tetrazole (0.88 grams,12.56 mmoles) for 24 hrs. The product was purified on silica gel byeluting the column with 100% ethyl acetate, giving 12.70 grams (89%yield). Fractions from the column were collected and evaporated to afoam. The resulting purified phosphinoamidite was analyzed by ³¹P NMR,giving a set of diastereomers at δ 121.7 and δ 122.0 ppm, and FAB MassSpectroscopy FAB+910 m/e (m+1), FAB−908 m/e (m−1).

EXAMPLE 30 Synthesis of 2′-O-(triisopropyl-silyloxymethyl)-3′-O-(diisopropylamino)-phosphino-acetic acid dimethyl-β-cyanoethylester-5′-O-di-p-anisylphenylmethyl-uridine

2′-O-(triisopropyl-silyloxymethyl)-5′-O-di-p-anisylphenylmethyl-uridinewas prepared from 5′-O-di-p-anisylphenylmethyl-uridine andchloromethoxytriisopropylsilane using the conditions described by Wagneret. al. (1974), J. Org. Chem. 39:24 and Pitsch et. al., WO. 99/09044,published Feb. 25, 1999. The protected nucleoside (5 grams, 6.8 mmol)was dissolved in anhydrous dichloromethane and reacted with acetic acid,(bis-(diisopropylamino) phosphino)-dimethylcyanoethyl ester (2.5 grams,6.8 mmoles), in the presence of tetrazole (0.38 grams, 5.44 mmoles) for24 hrs. The product was purified on silica gel by eluting the columnwith 50/50 hexanes/ethyl acetate giving 2.93 grams (43% yield).Fractions from the column were collected and evaporated to a foam. Theresulting purified phosphinoamidite was analyzed by ³¹P NMR, giving aset of diastereomers at δ 120.8 and δ 126.8 ppm, and FAB MassSpectroscopy FAB+1003 m/e (m+1), FAB−1001 m/e (m−1).

EXAMPLE 31 Synthesis of2′-O-methyl-3′-O-(diisopropylamino)-phosphino-acetic aciddimethyl-β-cyanoethyl ester-5′-O-di-p-anisylphenylmethyl-uridine

2′-O-methyl-5′-O-di-p-anisylphenylmethyl-uridine (10 grams, 18.3 mmol)was dissolved in anhydrous dichloromethane and reacted with acetic acid,(bis-(diisopropylamino) phosphino)-dimethylcyanoethyl ester (6.7 grams,18.3 mmoles), in the presence of tetrazole (1.02 grams, 14.6 mmoles) for24 hrs. The product was purified on silica gel by eluting the columnwith 50/50 hexanes/ethyl acetate, giving 8.80 grams (48% yield).Fractions from the column were collected and evaporated to a foam. Theresulting purified phosphinoamidite was analyzed by ³¹P NMR, giving aset of diastereomers at δ 123.6 and δ 125.9 ppm, and FAB MassSpectroscopy FAB+831 m/e (m+1), FAB−829 m/e (m−1).

EXAMPLE 32 Synthesis of thymidine-3′-O-acetic acid H-phosphonite

3′-O-(diisopropylamino)-phosphinoacetic acid dimethyl-β-cyanoethylester-5′-O-di-p-anisylphenylmethyl thymidine (1 gram, 1.2 mmol) wasdissolved in anhydrous acetonitrile (10 ml). Tetrazole (0.1 gram, 1.4mmol) was added to the solution along with 100 microliters of water. Theproduct was evaporated to dryness and purified by preparative HPLC on aC-18 column. The product was redissolved in a minimum volume ofacetonitrile and the dimethylcyanoethyl protecting group removed usingconcentrated ammonium hydroxide. The reaction mixture was evaporated todryness and the product repurified by preparative HPLC on a C-18 column.The di-p-anisylphenylmethyl protecting group was removed by redissolvingthe product in 80% acetic acid of 90 min. The acetic acid was dilutedwith water and the product purified by HPLC. FAB Mass SpectroscopyFAB+349 m/e (m+1), FAB−347 m/e (m−1).

EXAMPLE 33 General Method for Synthesis of PhosphonoacetateOligonucleotides

The chemical synthesis of phosphonoacetate oligonucleotides wasaccomplished using an ABI model 394 automated DNA synthesizer from PEBiosystems, Foster City Calif. The synthesis cycle used was adapted froma standard one micromolar β-cyanoethylphosphoramidite DNA synthesiscycle (FIG. 1). The coupling wait-time was increased to 1998 seconds.The oxidation step was accomplished prior to the capping step. The3′-O-(diisopropylamino)-phosphinoacetic acid dimethylcyanoethylester-5′-O-di-p-anisylphenylmethyl protected nucleosides were dissolvedin anhydrous acetonitrile at a concentration of 0.1M. The exocyclicamine groups were protected as follows: adenosine was protected by abenzoyl group; cytidine was protected with an acetyl group; andguanosine was protected with an isobutyryl group. Freshly sublimedtetrazole was used as an activator and was dissolved in anhydrousacetonitrile at a concentration of 0.45M. Trichloroacetic acid, 3%(wt/vol), dissolved in anhydrous dichloromethane, was used to deprotectthe 5′-O-di-p-anisylphenylmethyl groups prior to each round of coupling.Capping was accomplished using a two-part capping solution, designatedcap A and cap B. Cap A: 10% acetic anhydride in anhydroustetrahydrofuran. Cap B: 0.625% (wt/vol) 4-N,N-dimethylaminopyridine inanhydrous pyridine. Oxidation of the nascent internucleotide acetic acidphosphonite to the phosphonate was accomplished using(1S)-(+)(10-camphorsufonyl)-oxaziridine (CSO) dissolved in anhydrousacetonitrile at a concentration of 0.1 M. The oxidation wait time wasincreased to 180 seconds.

Post-synthesis, the control pore glass (CPG) was washed with anhydrousacetonitrile for 60 seconds and then flushed with a stream of argonuntil dry. The column was then fitted with two 1 mL syringes. The CPGwas exposed to a 1.5% 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) solution(15 μl in 1 mL of anhydrous acetonitrile) by pushing the solution backand forth with the syringe plungers. The reaction was allowed to proceedfor 30 minutes to remove the dimethylcyanoethyl groups. The DBU solutionwas removed and discarded. The CPG was then washed via syringe action inthe following manner: 20 mL anhydrous acetonitrile, followed by 2 mL of400 mM Tris-HCl. The CPG was flushed with argon until dry and thentransferred to a Teflon-lined screw-cap glass vial. The phosphonoacetateoligonucleotide was cleaved from the glass support, and the exocyclicamine-protecting groups were removed using a 40% solution of methylaminein water. Deprotection and cleavage were carried out at 55° C. for 15minutes and the vial was subsequently cooled to room temperature, andevaporated to dryness under vacuum.

EXAMPLE 34 General Method for Synthesis of PhosphonothioacetateOligonucleotides

The chemical synthesis of phosphonothioacetate oligonucleotides wasaccomplished using an ABI model 394 automated DNA synthesizer and thechemistry illustrated schematically in FIG. 2. The synthesis cycle usedwas adapted from the standard one micromolar β-cyanoethylphosphoramiditeDNA synthesis cycle. The coupling wait-time was increased to 2 times 999seconds. The oxidation step was accomplished prior to the capping step.The 3′-O-(diisopropylamino)-phosphinoacetic acid dimethylcyanoethylester-5′-O-di-p-anisylphenylmethyl protected nucleosides were dissolvedin anhydrous acetonitrile at a concentration of 0.1M. The exocyclicamine groups were protected as follows: adenosine was protected by abenzoyl group; cytidine was protected with an acetyl group; andguanosine was protected with an isobutyryl group. Freshly sublimedtetrazole was used as an activator and was dissolved in anhydrousacetonitrile at a concentration of 0.45 M. Trichloroacetic acid, 3%(wt/vol), dissolved in anhydrous dichloromethane, was used to deprotectthe 5′-O-di-p-anisylphenylmethyl groups prior to each round of coupling.Capping was accomplished using a two part capping solution, designatedcap A and cap B. Cap A: 10% acetic anhydride in anhydroustetrahydrofuran. Cap B: 0.625% (wt/vol) 4-N,N-dimethylaminopyridine inanhydrous pyridine. Oxidation of the nascent internucleotide acetic acidphosphonite to the thiophosphonate was accomplished using −1,1-dioxide3H-1, 2-benzodithiole-3-one-1,1-dioxide dissolved in anhydrousacetonitrile at a concentration of 0.05M. The oxidation wait time wasincreased to 60 seconds. Post-synthesis, the CPG was washed withanhydrous acetonitrile for 60 seconds and then flushed with a stream ofargon until dry. The column was then fitted with two 1 mL syringes. TheCPG was exposed to a 1.5% DBU solution (15 μl in 1 mL of anhydrousacetonitrile) by pushing the solution back and forth with the syringeplungers. The reaction was allowed to proceed for 30 minutes to removethe dimethylcyanoethyl groups. The DBU solution was removed anddiscarded. The CPG was then washed via syringe action in the followingmanner: 20 mL anhydrous acetonitrile, followed by 2 mL of 400 mMTris-HCl. The CPG was flushed with argon until dry and then transferredto a Teflon-lined, screw-cap glass vial. The phosphonothioacetateoligonucleotide was cleaved from the glass support, and the exocyclicamine-protecting groups were removed using a 40% solution of methylaminein water. Deprotection and cleavage were carried out at 55° C. for 15minutes and the vial was subsequently cooled to room temperature forfurther purification.

EXAMPLE 35 HPLC Purification of phosphonoacetate andthiophosphonoacetate oligonucleotides

Phosphonoacetate and thiophosphonoacetate oligonucleotides were purifiedby reverse-phase HPLC using the 5′-di-p-anisylphenylmethyl protectinggroup (trityl) for hydrophobic affinity. After cleavage from CPG, themethylamine solution was cooled to room temperature and filtered througha Pasteur pipette (fitted with a plug of glass wool) into a separatetube. The CPG was washed twice with 0.5 mL of water, filtered, and thecombined volumes were concentrated to dryness under vacuum. The crudeoligonucleotide mixture was dissolved in 0.5 mL of water forpurification by HPLC. Preparative HPLC utilized a Zorbax 300SB-C18column (9.4 mm ID×25 cm). Eluents were (a) 100 mM triethylammoniumacetate, pH 8.0, and (b) acetonitrile. The following gradient conditionswere used to elute the trityl-on oligonucleotides: 0-2 min, 8% B; 2-27minutes, 8 to 20% B; and finally 27-52 min, 20 to 80% B. The flow ratewas 1.2 mL/min. The desired fractions were collected, concentrated undervacuum, and dissolved in 100 μl of 10 mM Tris-HCl, pH 8. The tritylgroup was removed by treatment with 80% acetic acid (1 mL). After 1hour, the solution was concentrated to dryness and dissolved in 50 mMtriethylammonium acetate, pH 8 (0.5 mL). Finally, preparative HPLC,using the Zorbax 300SB-C18 column (9.4 mm ID×25 cm), was utilized toisolate and desalt the fully deprotected oligonucleotides. The elutionprofile used was as follows: 0% B, for 30 min; then a gradient of 0 to80% B, from 30 to 50 min. The flow rate was 1.2 mL/min.

EXAMPLE 36 Ion exchange HPLC analysis of 18-mer mixed sequencephosphonoacetate and phosphonothioacetate oligonucleotides

Mixed sequence 18-mer oligonucleotides 5′-CTCAAGTGGGCTGGTGAC-3′ weresynthesized using the synthesis cycles described for phosphonoacetates(Example 34, FIG. 1) and phosphonothioacetates (Example 35, FIG. 2) andpurified by reverse-phase HPLC. These sequences were analyzed forsynthetic yield of full-length product by ion exchange HPLC. The HPLCwas preformed using a Resource Q column (6.4 mm ID×30 mm) obtained fromAmersham/Pharmacia. Eluents were (a) 10 mM NaOH/80 mM NaBr and (b) 10 mMNaOH/1.5M NaBr. The gradient was 0% A to 100% B in 45 minutes at a flowrate of 1.5 mL/min. For both syntheses, per cycle coupling efficiency ofgreater than 97% per cycle was obtained. Results are shown in FIGS. 3and 4. Purified sequences were evaporated to dryness and redissolved inD₂0 for ³¹P NMR analysis of the phosphorus backbone using a VarianVXR-300 broadband NMR. Integration of the phosphorus signalsdemonstrated greater than 98% phosphonoacetate or phosphonothioacetateinternucleotide linkages in the purified products.

EXAMPLE 37 MALDI-TOF mass spectroscopy

Matrix-assisted laser desorption ionization time of flight (MALDI-TOF)spectroscopic analysis was performed on the mixed sequence 18-merphosphonoacetate and phosphonothioacetate oligonucleotides. Results areshown in FIGS. 5 and 6, respectively. The analysis was performed on aPerSeptive Biosystems Voyager Biospectrometry Workstation. Thecarboxylic acid modified oligonucleotides were concentrated to drynessand dissolved in isopropanol/water (1:1) to a final concentration of 200pmol/δl. Samples were prepared as described in the SequazymeOligonucleotide Sequencing Kit (PerSeptive Biosystems) with thefollowing modifications: 1 μl of oligonucleotide, 1 μl 25-mer DNAstandard, 1 μl of ammonium citrate buffer and 7 μl of matrix werecombined on a layer of parafilm coated with ammonium cation exchangebeads. After pipetting the solution over the beads for 60 seconds, 5 μlwas transferred to a gold-plated 100 well plate. The MALDI-TOFmeasurements were observed in the positive ion mode. The calculated massfor the mixed sequence 18-mer phosphonoacetate was 6270.2, and theobserved mass was 6271.5 (FIG. 5). The calculated mass for the mixedsequence 18-mer phosphonothioacetate was 6542.1, and the observed masswas 6541.2 (FIG. 6).

EXAMPLE 38 Evaluation of Nuclease Resistance of Phosphonoacetate andPhosphonothioacetate Oligonucleotides

This example describes an evaluation of the nuclease stabilities ofoligodeoxynucleotides (ODNs) containing phosphonoacetate andphosphonothioacetate internucleotide bonds. Nuclease resistance of thesemodified oligonucleotides was evaluated using Snake VenomPhosphodiesterase and DNase I, as follows.

ODN Synthesis and Purification: Control ODNs and phosphonoacetate ODNswere synthesized by solid-phase phoshoramidite methods on controlledpore glass using an automated synthesizer (Applied Biosystems model 394,Foster City, Calif.) substantially as described in Example 34.Phosphorothioate ODNs were prepared a similar manner using3H-1,2-benzodithiole-3-1,1 dioxide for sulfurization of the nascentphosphite internucleotide bonds (Glen Research, Sterling, Va.), asdescribed in Example 34, wherein the sulfurizing reagent was dissolvedin anhydrous acetonitrile (0.05 M) and the oxidation wait time was 45seconds. Phosphonoacetate and phosphonothioacetate ODNs were purified asdescribed in Examples 34 and 35. Commercially obtained phosphoramiditesynthons were used to incorporate a phosphodiester linkage on the 5′-endof the phosphonoacetate and phosphonothioacetate ODNs. Autoradiographicimaging was performed on a Molecular Dynamics Phosphorimager (Storm820).

Radiolabeling of Oligodeoxynucleotides: ODNs were 5′ end-labeled usingγ-³²P ATP and T4 polynucleotide kinase (Wu et al., “Purification andSequence Analysis of Synthetic Oligodeoxyribonucleotides,” inOligonucleotide Synthesis: A Practical Approach, Gait, Ed. (OxfordUniversity Press, 1990)). Phosphorylation of the phosphonoacetate andphosphonothioacetate ODNs was achieved using similar conditions and a 3to 4-fold increase in the number of equivalents of γ-³²P ATP. Theradiolabeled ODNs were purified by gel electrophoresis and ethanolprecipitation. Quantitation of radioactivity was accomplished usingImageQuant software (version 5.1).

MALDI-TOF mass spectroscopy. Matrix-assisted laser desorption ionizationtime of flight (MALDI-TOF) spectroscopic analysis was performed on aPerSeptive Biosystems Voyager Biospectrometry Workstation as describedin Example 37.

Exonuclease Stability: Exonuclease digestion experiments were carriedout using Phosphodiesterase I from Crotalus adamanteu (Cummings et al.(1996) Nucleic Acids Research 23:2019-2024). The assays were performedusing a mixture of oligonucleotides labeled with ³²P at the 5′-end(100,000 cpm) and unlabeled oligonucleotide (40 pmol) in a buffercontaining 50 mM Tris-HCl, pH 8.5, 72 mM NaCl and 14 mM MgCl₂. Enzymewas added to a final concentration of 0.5 units/mL (40 μl total reactionvolume). The reaction mixture was overlaid with 25 μl of mineral oil andincubated at 37° C. Aliquots (3.5 μl) were removed and quenched byadding 7M urea in TBE buffer (12 μl) and heating to 95° C. for 5minutes. Time points were taken at 0, 1, 3, 7, and 18 hours after theaddition of enzyme. Samples were stored at −70° C. until analysis byPAGE (20%, containing 7M urea).

Endonuclease Stability: Endonuclease digestion experiments were carriedout using DNase I (Boehringer-Mannheim). The assays were performed usinga mixture of oligodeoxynucleotide labeled with ³²P at the 5′-end(100,000 cpm) and unlabeled oligodeoxynucleotide (40 pmol) in a buffercontaining 40 mM Tris-HCl, pH 7.5, 6 mM MgCl₂. Slight excess of thecomplementary DNA strand (1.2 eq.) was added and the mixture was heatedto 95° C. for 5 minutes then chilled on ice for 30 minutes. Enzyme wasadded to a final concentration of 1 unit/μl (40 μl total reactionvolume). The reaction mixture was overlayed with 25 μl of mineral oiland incubated at 37° C. Aliquots (3.5 μl) were removed and quenched byadding 7M urea in TBE buffer (12 μl) and heating to 95° C. for 5minutes. Time points were taken at 0, 1, 3, 7, and 18 hours after theaddition of enzyme. Samples were stored at −70° C. until analysis byPAGE (20%, containing 7M urea).

Four 18-mer mixed sequence ODNs were synthesized, each having thesequence 5′-CTCAAGTGGGCTGGTGAC-3′, with one ODN containinginternucleotide phosphonoacetate linkages, one ODN containinginternucleotide phosphonothioacetate linkages, another ODN containinginternucleotide phosphorothioate linkages, and a fourth ODN containinginternucleotide phosphodiester linkages. The ODNs were synthesized,purified, and their identity verified by MALDI-TOF mass spectroscopyusing the above procedures. Results of the MALDI-TOF spectroscopicevaluation are set forth in Table 1: TABLE 1 Internucleotide BondCalculated Mass Observed Mass Phosphonoacetate 6227.9 6228.8Phosphodiester 5555.8 5555.1 Phosphonothioacetate 6525.9 6524.3Phosphorothioate 5827.8 5829.5The digestion reactions were monitored by radiolabeling the 5′-end ofthe ODN using γ-³²P ATP and T4 polynucleotide kinase. Attempts toradiolabel fully modified phosphonoacetate and phosphonothioacetate ODNswere unsuccessful using T4 Polynucleotide Kinase. In order tophosphorylate these modified ODNs, it was necessary to synthesize thesequences containing one phosphodiester internucleotide linkageimmediately 3′ to the 5′-end of the molecule. Phosphorylation of thesemodified ODNs required 4-fold excess ³²P-ATP with respect to theconditions used for unmodified DNA.

The phosphonoacetate, phosphonothioacetate, phosphorothioate, andphosphodiester ODNs were incubated with snake venom phosphodiesterase(SVP) and their degradation was monitored as a function of time.Aliquots were removed from the reaction mixture and quenched by additionof 7M urea followed by heating to 95° C. for 5 minutes. Each aliquot wasthen stored at −70° C. until analysis. Control experiments wereperformed without enzyme to compare full-length oligonucleotides toSVP-mediated hydrolysis products. The reaction products were separatedand analyzed by gel electrophoresis and imaged by autoradiography. FIG.7 shows the results observed for the ODNs when exposed to SVP. Nodetectable degradation products were observed for the reactionscontaining the phosphonoacetate ODN (“Ace,” lanes 2 through 5). Thephosphodiester ODN substrate was completely degraded within 60 minutes(“DNA,” lane 8). The phosphonothioacetate ODN gave no detectablehydrolysis products through the course of the experiment (“S-Ace,” lanes14 through 17). Lanes 20 through 23 show the hydrolysis products for thephosphorothioate substrate (“S1”) upon incubation with SVP. After 60minutes, approximately 65% of the full-length ODN was remained, andafter 18 hours 40% full-length ODN remained.

To characterize the phosphonoacetate and phosphonothioacetate ODNs'stability towards endonuclease, the radiolabeled modified ODNs werehybridized to complementary sequences of unmodified ODNs and incubatedwith DNase I. Duplexes were pre-formed by mixing the various ODNsseparately with a non-radiolabeled complementary 18-mer DNA strand. Themixtures were heated to 95° C. for 5 minutes followed by slow coolingand incubation on ice for 30 minutes. The mixture was equilibrated toroom temperature, DNase I added, and the reaction monitored as afunction of time. Control experiments were performed without enzyme tocompare full-length oligonucleotides to DNase I-mediated hydrolysisproducts. The reaction products were resolved by gel electrophoresis andimaged by autoradiography. FIG. 8 shows the typical results of exposureto DNase I for the 18-mer duplexes tested. Analysis of lanes 2 through 5show no degradation products for the phosphonoacetate (“Ace”)oligodeoxynucleotide substrate upon incubation with DNase I, even afterexposure to the enzyme for 18 hours. However, analysis of lane 8 (“DNA”)shows that the full-length DNA substrate was completely degraded within1 hour and that several hydrolysis fragments were consistently observedduring the assay. Alternatively, evaluation of lanes 14 through 17reveal that no hydrolysis products were observed for thephosphonothioacetate (“S-Ace”) analog during the time-course of theexperiment. For the phosphorothioate substrate (“S1,” lanes 20 through23) some hydrolysis products appeared through out the time course of theexperiment (approximately 12% degraded over 18 hours).

The results indicate that phosphonoacetate andphosphonothioacetate-containing oligodeoxynucleotides are highlynuclease-resistant.

EXAMPLE 39 Evaluation of Phosphonoacetate and PhosphonothioacetateOligonucleotide-RNA Duplexes as Substrates for RNaseH

This example evaluates the ability of phosphonoacetate andphosphonothioacetate ODNs to bind to complementary RNA and subsequentlyact as substrates for E. coli RNaseH1, i.e., directing the hydrolysis ofcomplementary RNA in the presence of the RnaseH. Four ODNs weresynthesized as in the preceding example, i.e., 18-mer mixed sequenceODNs each having the sequence 5′-CTCAAGTGGGCTGGTGAC-3′, with one ODNcontaining internucleotide phosphonoacetate linkages, one ODN containinginternucleotide phosphonothioacetate linkages, another ODN containinginternucleotide phosphorothioate linkages, and a fourth ODN containinginternucleotide phosphodiester linkages. The ODNs were purified, andtheir identity verified by MALDI-TOF mass spectroscopy using theprocedures of the preceding example.

Hybridization of oligonucleotides to complementary RNA: Hybridizationstudies were performed using equimolar amounts (1 μM) of thephosphonoacetate, phosphonothioacetate, phosphorothioate, 2′-O-methylmodified or phosphodiester oligonucleotides with the complementaryoligoribonucleotide strand. Experiments were performed using twodifferent buffer conditions: 1) 1M sodium chloride in phosphate-bufferedsaline; and 2) RNaseH buffer conditions.

Melting Point Measurements: Melting points, T_(m)s, for the 18-merheteroduplexes were determined on a Varian Cary 1E UV-visiblespectrometer. The absorbance at 260 nm was measured while thetemperature of the sample was increased at rate of 1.0° C./min.Phosphonoacetate, phosphonothioacetate, phosphorothioate, 2′-O-Methylmodified, and phosphodiester oligonucleotides were separately mixed withcomplementary RNA in a 1 mL cuvette and the T_(m) determined as themaximum of the first derivative of the melting curve. Typicalconcentrations were 1 μM in each strand, pH 7.2 and contained 1 mM EDTA,10 mM Na₂HPO₄+1.0M NaCl. Melting curves were also determined using E.coli RNaseH1 buffer conditions: 20 mM HEPES-KOH (pH 7.8), 50 mM KCl, 10mM MgCl₂, 1 mM DTT.

Hydrolysis ofRNA hetero-duplexes with E. coli RnaseH1. Complementary RNAwas 5′ end-labeled using γ-³²P ATP and T4 polynucleotide kinase (Wu etal., supra). A mixture of 5′³²P labeled RNA (100,000 cpm/reaction) andunlabeled RNA (125 pmol) were mixed with 100 pmol of complementaryoligonucleotide in a buffer containing 20 mM HEPES-KOH (pH 7.8), 50 mMKCl, 10 mM MgCl₂, 1 mM DTT, and 40 units of RNasin (Promega) andincubated at 37° C. for 60 minutes. Two units of E. coli RNaseH1(Promega) were added, and the reaction was allowed to proceed for fourhours at 37° C. (20 μl total reaction volume). Aliquots of the reactionmixture (3.5 μl) were quenched with 7M urea in TBE buffer (12 μl) andstored at −70° C. until analysis by gel electrophoresis (20%, 19:1crosslink).

RNaseH1 Activity of Phosphonoacetate and Phosphonothioacetate-RNAheteroduplexes: The RNaseH experiments were monitored by radiolabelingthe 5′-end of the RNA strand using γ-³²P ATP and T4 polynucleotidekinase (Wu et al., supra). Phosphonoacetate, phosphonothioacetate,phosphorothioate, 2′-O-methyl sugar-modified phosphodiester andphosphodiester oligonucleotides were separately mixed with complementaryRNA and allowed to equilibrate at 37° C. for one hour. Reactions wereinitiated by the addition of RNaseH and incubated at 37° C. for fourhours. Aliquots were removed from the reaction mixture and quenched byaddition of 7M urea in TBE buffer followed by heating to 95° C. for 5minutes. Each aliquot was then stored on ice until analysis. Thereaction products were resolved by gel electrophoresis and imaged byautoradiography. Results from a typical RNaseH1 experiment are shown inFIG. 9. Each oligonucleotide was tested in the presence and absence ofthe enzyme. Examination of lane 4 shows the hydrolysis of complementaryRNA when hybridized to the phosphonoacetate ODN in the presence of E.coli RNaseH1. The RNA was degraded by approximately 66% during thecourse of the experiment. Under the same conditions, no hydrolysisproducts were observed when the RNA was incubated with non-complementaryphosphonoacetate ODN (lane 14). When exposed to RNaseH1 approximately67% of the RNA was degraded in the presence of complementaryphosphonothioacetate ODN (lane 8). Furthermore, the presence of thenon-complementary phosphonothioacetate ODN did not result in anysignificant hydrolysis products when incubated with RNaseH1 (lane 16).When the phosphodiester ODN-RNA and phosphorothioate ODN-RNA duplexeswere incubated with RNaseH1 nearly 100% of the starting material wasconverted to hydrolysis products. In the presence of complementary2′-O-methyl oligoribonucleotide, no RNA hydrolysis products weredetected. The migration patterns of the radiolabeled RNA in the2′-O-methyl modified-RNA lanes were congruent with duplexoligonucleotide structures as assessed by native gel electrophoresis ofthe heteroduplexes. No hydrolysis products were generated in the absenceof a complementary oligonucleotide strand (lane 2).

1. A compound having the structural formula (I)

wherein: R¹ is hydrogen, a protecting group removable by an eliminationreaction, hydrocarbyl, substituted hydrocarbyl, heteroatom-containinghydrocarbyl or substituted heteroatom-containing hydrocarbyl; R² and R³are independently selected from the group consisting of hydrocarbyl,substituted hydrocarbyl, heteroatom-containing hydrocarbyl andsubstituted heteroatom-containing hydrocarbyl, or R² and R³ are linkedto form a substituted or unsubstituted, five- or six-memberednitrogen-containing heterocycle; R⁴ is NR⁵R⁶, halogen or DL, wherein R⁵and R⁶ are independently selected from the group consisting ofhydrocarbyl, substituted hydrocarbyl, heteroatom-containing hydrocarbyland substituted heteroatom-containing hydrocarbyl, or R⁵ and R⁶ arelinked to form a substituted or unsubstituted, five- or six-memberednitrogen-containing heterocycle, D is O, S or NH, and L is aheteroatom-protecting group, unsubstituted hydrocarbyl, substitutedhydrocarbyl, heteroatom-containing hydrocarbyl, or substitutedheteroatom-containing hydrocarbyl; X is O, S NH or NR⁷ wherein R⁷ ishydrocarbyl, substituted hydrocarbyl, heteroatom-containing hydrocarbylor substituted heteroatom-containing hydrocarbyl; n is zero or 1; Y is—(Y′)_(m)—(CR⁸R⁹)— wherein m is zero or 1, Y′ is hydrocarbylene,substituted hydrocarbylene, heteroatom-containing hydrocarbylene, orsubstituted heteroatom-containing hydrocarbylene, wherein R⁸ and R⁹ areindependently selected from the group consisting of hydrogen,hydrocarbyl, substituted hydrocarbyl, heteroatom-containing hydrocarbyland substituted heteroatom-containing hydrocarbyl, with the proviso thatwhen n is 1, m is zero and R⁸ and R⁹ are not both hydrogen, then R¹ iseither hydrogen or a protecting group removable by an eliminationreaction; and Z is O, S, NH or NR¹⁰ wherein R¹⁰ is as defined for R⁷. 2.A compound having the structural formula (I)

wherein: R¹ is hydrogen, a protecting group removable by an eliminationreaction, or an unsubstituted, substituted, heteroatom-containing orsubstituted heteroatom-containing moiety selected from the groupconsisting of alkyl, aryl, aralkyl, alkaryl, cycloalkyl,cycloalkylalkyl, cycloalkylaryl, alkenyl, cycloalkenyl, alkynyl andaralkynyl; R² and R³ are unsubstituted, substituted,heteroatom-containing or substituted heteroatom-containing moietiesselected from the group consisting of alkyl, aryl, aralkyl, alkaryl,cycloalkyl, cycloalkylalkyl, cycloalkylaryl, alkenyl, cycloalkenyl,alkynyl and aralkynyl, or R² and R³ are linked to form a substituted orunsubstituted, five- or six-membered nitrogen-containing heterocycle; R⁴is NR⁵R⁶ wherein R⁵ and R⁶ are as defined for R² and R³; X is O or S; nis zero or 1; Y is —(Y′)_(m)—(CR⁸R⁹)— wherein m is zero or 1, Y′ ishydrocarbylene, substituted hydrocarbylene, heteroatom-containinghydrocarbylene, or substituted heteroatom-containing hydrocarbylene,wherein R⁸ and R⁹ are as defined for R¹, with the proviso that when n is1, m is zero and R⁸ and R⁹ are both hydrogen, then R¹ is either hydrogenor a protecting group removable by an elimination reaction; and Z is Oor S.
 3. The compound of claim 2, wherein n is zero.
 4. The compound ofclaim 2, wherein n is
 1. 5. The compound of claim 4, wherein m is zero.6. The compound of claim 4, wherein m is
 1. 7. The compound of claim 2,wherein Z is O.
 8. The compound of claim 7, wherein X is O.
 9. Thecompound of claim 2, wherein R¹ is a protecting group removable by anelimination reaction.
 10. The compound of claim 9, wherein R¹ isselected from the group consisting of β-cyanoethyl, methyl-β-cyanoethyl,dimethyl-β-cyanoethyl, phenylsulfonylethyl, methyl-sulfonylethyl,p-nitrophenylsulfonylethyl, 2,2,2-trichloro-1,1-dimethylethyl,2-(4-pyridyl)ethyl, 2-(2-pyridyl)ethyl, allyl,4-methylene-l-acetylphenol, β-thiobenzoylethyl,1,1,1,3,3,3-hexafluoro-2-propyl, 2,2,2-trichloroethyl,p-nitrophenylethyl, p-cyanophenyl-ethyl, 9-fluorenylmethyl,1,3-dithionyl-2-methyl, 2-(trimethylsilyl)ethyl, 2-methylthioethyl,2-(diphenylphosphino)ethyl, 1-methyl-1-phenylethyl, 3-buten-1-yl,4-(trimethylsilyl)-2-buten- 1-yl, cinnamyl, α-methylcinnamyl, and8-quinolyl.
 11. The compound of claim 2, wherein R¹ is hydrogen.
 12. Thecompound of claim 2, wherein NR²R³ and NR⁵R⁶ are independently selectedfrom the group consisting of dimethylamino, diethylamino,diisopropylamino, dibutylamino, methylpropylamino, methylhexylamino,methylcyclohexylamino, ethylcyclopropylamino, ethylchloroethylamino,methylbenzylamino, methylphenylamino, thiomorpholino, methyltoluylamino,methyl-p-chlorophenylamino, methylcyclohexylamino,bromobutylcyclohexylamino, methyl-p-cyanophenylamino,ethyl-β-cyanoethylamino, piperidino, 2,6,-dimethylpiperidino,pyrrolidino, piperazino, isopropylcyclohexylamino, and morpholino. 13.The compound of claim 12, wherein NR²R³ and NR⁵R⁶ are diisopropylamino.14. The compound of claim 1, wherein R⁴ is DL.
 15. The compound of claim14, wherein L is a heteroatom-protecting group removable by anelimination reaction.
 16. The compound of claim 15, wherein L isselected from the group consisting of β-cyanoethyl, methyl-β-cyanoethyl,dimethyl-β-cyanoethyl, phenylsulfonylethyl, methyl-sulfonylethyl,p-nitrophenylsulfonylethyl, 2,2,2-trichloro-1,1-dimethylethyl,2-(4-pyridyl)ethyl, 2-(2-pyridyl)ethyl, allyl,4-methylene-1-acetylphenol, β-thiobenzoylethyl,1,1,1,3,3,3-hexafluoro-2-propyl, 2,2,2-trichloroethyl,p-nitrophenylethyl, p-cyanophenyl-ethyl, 9-fluorenylmethyl,1,3-dithionyl-2-methyl, 2-(trimethylsilyl)ethyl, 2-methylthioethyl,2-(diphenylphosphino)ethyl, 1-methyl-1-phenylethyl, 3-buten-1-yl,4-(trimethylsilyl)-2-buten-1-yl, cinnamyl, α-methylcinnamyl and8-quinolyl.
 17. A compound having the structural formula (II)

wherein: R¹ is hydrogen, lower alkyl, or an electron-withdrawingβ-substituted aliphatic group; R² and R³ are lower alkyl, or R² and R³are linked to form a piperidino, piperazino or morpholino ring; R⁴ isNR⁵R⁶, chloro or OL wherein R⁵ and R⁶ are as defined for R² and R³, andL is selected from the group consisting of β-cyanoethyl,methyl-β-cyanoethyl, dimethyl-β-cyanoethyl, phenylsulfonylethyl,methyl-sulfonylethyl, p-nitrophenylsulfonylethyl,2,2,2-trichloro-1,1-dimethylethyl, 2-(4-pyridyl)ethyl,2-(2-pyridyl)ethyl, allyl, 4-methylene-1-acetylphenol,β-thiobenzoylethyl, 1,1,1,3,3,3-hexafluoro-2-propyl,2,2,2-trichloroethyl, p-nitrophenylethyl, p-cyanophenyl-ethyl,9-fluorenylmethyl, 1,3-dithionyl-2-methyl, 2-(trimethylsilyl)ethyl,2-methylthioethyl, 2-(diphenylphosphino)ethyl, 1-methyl-1-phenylethyl,3-buten-1-yl, 4-(trimethylsilyl)-2-buten-1-yl, cinnamyl,α-methylcinnamyl and 8-quinolyl; n is zero or 1; and Y is—(Y′)_(m)—(CH₂)— wherein m is zero or 1 and Y′ is lower alkylene, withthe proviso that when n is 1 and m is zero, then R¹ is either hydrogenor an electron-withdrawing β-substituted aliphatic group.
 18. Anoligonucleotide containing at least one internucleotide linkage havingthe structure of formula (VIII)

wherein: R¹ is hydrogen, a protecting group removable by an eliminationreaction, hydrocarbyl, substituted hydrocarbyl, heteroatom-containinghydrocarbyl or substituted heteroatom-containing hydrocarbyl; X is O, SNH or NR⁷ wherein R⁷ is hydrocarbyl, substituted hydrocarbyl,heteroatom-containing hydrocarbyl or substituted heteroatom-containinghydrocarbyl; n is zero or 1; Y is —(Y′)_(m)— (CR⁸R⁹)— wherein m is zeroor 1, Y′ is hydrocarbylene, substituted hydrocarbylene,heteroatom-containing hydrocarbylene, or substitutedheteroatom-containing hydrocarbylene, wherein R⁸ and R⁹ areindependently selected from the group consisting of hydrogen,hydrocarbyl, substituted hydrocarbyl, heteroatom-containing hydrocarbyland substituted heteroatom-containing hydrocarbyl; and Z is O, S, NH orNR¹⁰ wherein R¹⁰ is as defined for R⁷.
 19. An oligonucleotide containingat least one internucleotide linkage having the structure of formula(VIII)

wherein: R¹ is hydrogen, a protecting group removable by an eliminationreaction, or an unsubstituted, substituted, heteroatom-containing orsubstituted heteroatom-containing moiety selected from the groupconsisting of alkyl, aryl, aralkyl, alkaryl, cycloalkyl,cycloalkylalkyl, cycloalkylaryl, alkenyl, cycloalkenyl, alkynyl andaralkynyl; X is O or S; n is zero or 1; Y is —(Y′)_(m)—(CR⁸R⁹)— whereinm is zero or 1, Y′ is an unsubstituted, substituted,heteroatom-containing or substituted heteroatom-containing moietyselected from the group consisting of alkylene, arylene, aralkylene,alkarylene, cycloalkylene, cycloalkylalkylene, cycloalkylarylene,alkenylene, cycloalkenylene, alkynylene and aralkynylene, wherein R⁸ andR⁹ are independently selected from hydrogen and unsubstituted,substituted, heteroatom-containing or substituted heteroatom-containingmoieties selected from the group consisting of alkyl, aryl, aralkyl,alkaryl, cycloalkyl, cycloalkylalkyl, cycloalkylaryl, alkenyl,cycloalkenyl, alkynyl and aralkynyl; and Z is O or S.
 20. Theoligonucleotide of claim 19, wherein n is zero.
 21. The oligonucleotideof claim 19, wherein n is
 1. 22. The oligonucleotide of claim 21,wherein m is zero.
 23. The oligonucleotide of claim 21, wherein m is 1.24. The oligonucleotide of claim 19, wherein R¹ is a protecting groupremovable by an elimination reaction.
 25. The oligonucleotide of claim24, wherein R¹ is selected from the group comprised of β-cyanoethyl,methyl-β-cyanoethyl, dimethyl-β-cyanoethyl, phenylsulfonylethyl,methyl-sulfonylethyl, p-nitrophenylsulfonylethyl,2,2,2-trichloro-1,1-dimethylethyl, 2-(4-pyridyl)ethyl,2-(2-pyridyl)ethyl, allyl, 4-methylene-1-acetylphenol,β-thiobenzoylethyl, 1,1,1,3,3,3-hexafluoro-2-propyl,2,2,2-trichloroethyl, p-nitrophenylethyl, p-cyanophenyl-ethyl,9-fluorenylmethyl, 1,3-dithionyl-2-methyl, 2-(trimethylsilyl)ethyl,2-methylthioethyl, 2-(diphenylphosphino)ethyl, 1-methyl-1-phenylethyl,3-buten-1-yl, 4-(trimethylsilyl)-2-buten-1-yl, cinnamyl,α-methylcinnamyl, and 8-quinolyl.
 26. The oligonucleotide of claim 19,wherein R¹ is hydrogen.
 27. An oligonucleotide containing at least oneinternucleotide linkage having the structure of formula (IX)

wherein: R¹ is hydrogen, lower alkyl, or a hydroxyl-protecting groupremovable by an elimination reaction; n is zero or 1; and Y is—(Y′)_(m)—(CH₂)— wherein m is zero or 1 and Y′ is lower alkylene.
 28. Anoligonucleotide containing at least one internucleotide linkage havingthe structure of formula (X)

wherein: R¹ is hydrogen, a protecting group removable by an eliminationreaction, or an unsubstituted, substituted, heteroatom-containing orsubstituted heteroatom-containing moiety selected from the groupconsisting of alkyl, aryl, aralkyl, alkaryl, cycloalkyl,cycloalkylalkyl, cycloalkylaryl, alkenyl, cycloalkenyl, alkynyl andaralkynyl; X is O, S NH or NR⁷ wherein R⁷ is hydrocarbyl, substitutedhydrocarbyl, heteroatom-containing hydrocarbyl or substitutedheteroatom-containing hydrocarbyl; n is zero or 1; Y is—(Y′)_(m)—(CR⁸R⁹)— wherein m is zero or 1, Y′ is hydrocarbylene,substituted hydrocarbylene, heteroatom-containing hydrocarbylene, orsubstituted heteroatom-containing hydrocarbylene, wherein R⁸ and R⁹ areindependently selected from the group consisting of hydrogen,hydrocarbyl, substituted hydrocarbyl, heteroatom-containing hydrocarbyland substituted heteroatom-containing hydrocarbyl; and Z is O, S, NH orNR¹⁰ wherein R¹⁰ is as defined for R⁷.
 29. An oligonucleotide containingat least one internucleotide linkage having the structure of formula (X)

wherein: R¹ is hydrogen, a protecting group removable by an eliminationreaction, or an unsubstituted, substituted, heteroatom-containing orsubstituted heteroatom-containing moiety selected from the groupconsisting of alkyl, aryl, aralkyl, alkaryl, cycloalkyl,cycloalkylalkyl, cycloalkylaryl, alkenyl, cycloalkenyl, alkynyl andaralkynyl; X is O or S; n is zero or 1; Y is —(Y′)_(m)—(CR⁸R⁹)— whereinm is zero or 1, Y′ is an unsubstituted, substituted,heteroatom-containing or substituted heteroatom-containing moietyselected from the group consisting of alkylene, arylene, aralkylene,alkarylene, cycloalkylene, cycloalkylalkylene, cycloalkylarylene,alkenylene, cycloalkenylene, alkynylene and aralkynylene, wherein R⁸ andR⁹ are independently selected from hydrogen and unsubstituted,substituted, heteroatom-containing or substituted heteroatom-containingmoieties selected from the group consisting of alkyl, aryl, aralkyl,alkaryl, cycloalkyl, cycloalkylalkyl, cycloalkylaryl, alkenyl,cycloalkenyl, alkynyl and aralkynyl; and Z is O or S.
 30. Theoligonucleotide of claim 29, wherein n is zero.
 31. The oligonucleotideof claim 29, wherein n is
 1. 32. The oligonucleotide of claim 31,wherein m is zero.
 33. The oligonucleotide of claim 29, wherein m is 1.34. The oligonucleotide of claim 29, wherein R¹ is a protecting groupremovable by an elimination reaction.
 35. The oligonucleotide of claim34, wherein R¹ is selected from the group comprised of β-cyanoethyl,methyl-β-cyanoethyl, dimethyl-β-cyanoethyl, phenylsulfonylethyl,methyl-sulfonylethyl, p-nitrophenylsulfonylethyl,2,2,2-trichloro-1,1-dimethylethyl, 2-(4-pyridyl)ethyl,2-(2-pyridyl)ethyl, allyl, 4-methylene-1-acetylphenol,β-thiobenzoylethyl, 1,1,1,3,3,3-hexafluoro-2-propyl,2,2,2-trichloroethyl, p-nitrophenylethyl, p-cyanophenyl-ethyl,9-fluorenylmethyl, 1,3-dithionyl-2-methyl, 2-(trimethylsilyl)ethyl,2-methylthioethyl, 2-(diphenylphosphino)ethyl, 1-methyl-1-phenylethyl,3-buten-1-yl, 4-(trimethylsilyl)-2-buten-1-yl, cinnamyl,α-methylcinnamyl, and 8-quinolyl.
 36. The oligonucleotide of claim 29,wherein R¹ is hydrogen.
 37. An oligonucleotide containing at least oneinternucleotide linkage having the structure of formula (XI)

wherein: R¹ is hydrogen, lower alkyl, or a hydroxyl-protecting groupremovable by an elimination reaction; n is zero or 1; and Y is—(Y′)_(m)—(CH₂)— wherein m is zero or 1 and Y′ is lower alkylene.
 38. Amethod for phosphitylating a nucleoside or an oligonucleotide having afree 3′ or 5′ hydroxyl moiety, comprising contacting the nucleoside oroligonucleotide with the compound of claim 1 under conditions effectiveto convert the free hydroxyl moiety to a phosphino substituent,providing a phosphitylated product.
 39. The method of claim 38, whereinthe free hydroxyl moiety is a 5′ hydroxyl moiety.
 40. The method ofclaim 38, wherein the nucleoside or oligonucleotide is bound to a solidsupport.
 41. The method of claim 38, further comprising reacting thephosphitylated product with an acidic compound effective to convert thephosphitylated product to a nucleoside or oligonucleotide H-phosphonite.42. In a method for phosphitylating a compound having a nucleophilicmoiety using a phosphitylating reagent, the improvement which comprisesusing as the phosphitylating reagent the compound of claim 1.